Double-stranded oligonucleotide, composition and conjugate comprising double-stranded oligonucleotide, preparation method thereof and use thereof

ABSTRACT

Provided is a modified double-stranded oligonucleotide, in which the sense strand comprises a nucleotide sequence 1, the anti-sense strand comprises a nucleotide sequence 2, the nucleotide sequences 1 and 2 are both 19 nucleotides in length, and in the direction from 5′ end to 3′ end, nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 and nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence 2 are all fluoro-modified nucleotides, and each nucleotide at other positions is independently one of non-fluoro-modified nucleotides. Further provided are a pharmaceutical composition and a conjugate comprising the oligonucleotide, and pharmaceutical use thereof.

CROSS REFERENCE OF RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/758,720, filed on Apr. 23, 2020, entitled “DOUBLE-STRANDEDOLIGONUCLEOTIDE, COMPOSITION AND CONJUGATE COMPRISING DOUBLE-STRANDEDOLIGONUCLEOTIDE, PREPARATION METHOD THEREOF AND USE THEREOF,” which inturn is a national stage application of PCT/CN2018/118212, filed on Nov.29, 2018, which claims priority to Chinese Patent Application No.201711249356.9, filed on Dec. 1, 2017, Chinese Patent Application No.201711249345.0, filed on Dec. 1, 2017, Chinese Patent Application No.201711249333.8, filed on Dec. 1, 2017, Chinese Patent Application No.201711479058.9, filed on Dec. 29, 2017, Chinese Patent Application No.201810951752.4, filed on Aug. 21, 2018, and Chinese Patent ApplicationNo. 201811165363.5, filed on Sep. 30, 2018. The entire content of eachof the prior applications is hereby incorporated by reference.

SEQUENCE LISTING

Incorporated by reference herein in its entirety is a computer-readablesequence listing submitted via EFS-Web and identified as follows: One(87534 byte ASCII (Text)) file named “20211012RB069PCT-ESP1V192724ZX-CNSZRB-US-updated.txt” created on Oct. 12, 2021.

BACKGROUND OF THE INVENTION

The use of double-stranded oligonucleotides as pharmaceutical activeingredients has been well-known to the public. Delivery system is one ofkey technologies in the development of small RNA drugs. One type ofsmall RNA delivery system is a targeted conjugation delivery technologyfor liver cells.

SUMMARY OF THE INVENTION

In some embodiments, the present disclosure provides a double-strandedoligonucleotide comprising a sense strand and an antisense strand, eachnucleotide in the sense strand and the antisense strand being a modifiednucleotide, wherein the sense strand comprises a nucleotide sequence 1,and the antisense strand comprises a nucleotide sequence 2; thenucleotide sequence 1 and the nucleotide sequence 2 are both 19nucleotides in length and are at least partly reverse complementary toform a double-stranded complementary region; the nucleotide sequence 2is at least partly reverse complementary to a first nucleotide sequencesegment, which refers to a segment of nucleotide sequence in the targetmRNA; in the direction from 5′ terminal to 3′ terminal, the nucleotidesat positions 7, 8 and 9 of the nucleotide sequence 1 are fluoro modifiednucleotides, and each of the nucleotides at the other positions in thenucleotide sequence 1 is independently a non-fluoro modified nucleotide;the first nucleotide at 5′ terminal of the nucleotide sequence 2 is thefirst nucleotide at 5′ terminal of the antisense strand, the nucleotidesat positions 2, 6, 14 and 16 of the nucleotide sequence 2 are fluoromodified nucleotides, and each of the nucleotides at the other positionsin the nucleotide sequence 2 is independently a non-fluoro modifiednucleotide.

In some embodiments, the present disclosure further provides apharmaceutical composition comprising the double-strandedoligonucleotide of the present disclosure and a pharmaceuticallyacceptable carrier.

In some embodiments, the present disclosure further provides a conjugatecomprising the double-stranded oligonucleotide the present disclosureand a ligand conjugated to the double-stranded oligonucleotide.

In some embodiments, the present disclosure provides use of thedouble-stranded oligonucleotide, pharmaceutical composition or conjugateof the present disclosure in the manufacture of a medicament fortreating and/or preventing a pathological condition or disease caused bythe expression of a specific gene in hepatocytes.

In some embodiments, the present disclosure provides a method fortreating a pathological condition or disease caused by the expression ofa specific gene in hepatocytes, comprising administering thedouble-stranded oligonucleotide, pharmaceutical composition or conjugateof the present disclosure, to a subject suffering from such a disease.

In some embodiments, the present disclosure provides is a method forinhibiting the expression of a specific gene in hepatocytes, comprisingcontacting the hepatocytes with the double-stranded oligonucleotide,pharmaceutical composition or conjugate of the present disclosure.

In some embodiments, the present disclosure provides is a kit comprisingthe double-stranded oligonucleotide, pharmaceutical composition orconjugate of the present disclosure.

Additional features and advantages of the present invention will beillustrated in detail in the following specific embodiments.

INCORPORATION BY REFERENCE

All publications, patents and patent applications mentioned in thisdescription are herein incorporated by reference to the same extent asif each individual publication, patent and patent application werespecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show the semiquantitative result of the stability test ofthe siRNA conjugates in the Tritosome in vitro.

FIGS. 3 and 4 show the semiquantitative result of the stability test ofthe siRNA conjugates in human plasma in vitro.

FIGS. 5 and 6 show the semiquantitative result of the stability test ofthe siRNA conjugates in monkey plasma in vitro.

FIGS. 7-10 are metabolic curves over time showing PK/TK plasma or tissueconcentration for: Conjugate A1 in rat plasma at a dosage of 10 mg/kg(FIG. 7 ); Conjugate A1 in rat liver and kidney at a dosage of 10 mg/kg(FIG. 8 ); Conjugate A1 in rat plasma at a dosage of 50 mg/kg (FIG. 9 );Conjugate A1 in rat liver and kidney at a dosage of 50 mg/kg (FIG. 10 ).

FIGS. 11-14 respectively show inhibitory effect of the conjugates of thepresent disclosure on HBV mRNA in 44BriHBV models.

FIGS. 15-16 respectively show inhibitory effect over time of theconjugates of the present disclosure on serum HBsAg and HBV DNA inAAV-HBV models.

FIG. 17 shows inhibitory effect over time of the conjugates of thepresent disclosure on serum HBsAg in M-Tg models.

FIGS. 18-19 respectively show inhibitory effect over time of theconjugates of the present disclosure on serum HBsAg and HBV DNA in 1.28copy HBV-Tg models.

FIGS. 20A, 20B, 20C, and 20D respectively show inhibitory effect ofConjugate A1 at different concentrations on the expression of GSCM,GSSM, PSCM and PSSM.

FIGS. 21-22 respectively show in intro inhibitory effect of theconjugates of the present disclosure on target mRNA and off-target mRNA.

FIGS. 23-25 respectively show the results of the stability tests of theconjugates of the present disclosure in vitro.

FIGS. 26-28 show inhibitory effect of the conjugates of the presentdisclosure on HBV mRNA in vivo.

FIGS. 29-31 show inhibitory effect over time of the conjugates of thepresent disclosure on the expression of HBsAg and HBV DNA in the sera ofdifferent HBV transgenic mice.

FIGS. 32-34 respectively show the results of the stability tests of theconjugates of the present disclosure in vitro.

FIGS. 35-36 show in intro inhibitory effect of the conjugates of thepresent disclosure on target mRNA and off-target mRNA.

FIG. 37 shows in vivo inhibitory effect of the conjugates of the presentdisclosure on mRNA in 44BriHBV models.

FIG. 38 shows inhibitory effect over time of the conjugates of thepresent disclosure on the expression of HBsAg in mice.

FIG. 39 shows in vivo inhibitory effect of the conjugates of the presentdisclosure on mRNA in M-Tg mouse models.

FIGS. 40-42 show the results of the stability tests of the conjugates ofthe present disclosure in vitro.

FIGS. 43-44 show in intro inhibitory effect of the conjugates of thepresent disclosure on target mRNA and off-target mRNA.

FIG. 45 shows in vivo inhibitory effect of the conjugates of the presentdisclosure on HBV mRNA.

FIG. 46 shows inhibitory effect over time of the conjugates of thepresent disclosure on HBsAg expression in HBV transgenic mice serum.

FIG. 47 shows inhibitory effect of the conjugates of the presentdisclosure on HBV mRNA in M-Tg mouse models.

FIGS. 48A-48D show inhibitory effect of comparative siRNA3 on targetmRNA and off-target mRNA in intro.

FIGS. 49A-49D show inhibitory effect of the siRNA E1 of the presentdisclosure on target mRNA and off-target mRNA in intro.

FIGS. 50A-50B respectively show inhibitory effect of the siRNA and siRNAconjugates of the present disclosure on ANGPTL3 mRNA in intro.

FIGS. 51A-51D respectively show the results of the stability tests ofthe conjugates of the present disclosure in vitro.

FIGS. 52A-52D show inhibition percentages of the conjugates of thepresent disclosure against blood lipid, represented by total cholesterol(CHO) and triglyceride (TG) in serum.

FIGS. 53A-53D show inhibition percentages of the conjugates of thepresent disclosure against ANGPTL3 mRNA in vivo.

FIGS. 54A-54D respectively show inhibition percentages of the conjugatesof the present disclosure against blood lipid, represented by totalcholesterol (CHO) and triglyceride (TG) in serum.

FIGS. 55A and 55B show inhibition percentages over time of theconjugates of the present disclosure against blood lipid, represented bytotal cholesterol (CHO) and triglyceride (TG) in serum; and FIG. 55Cshows the inhibition percentage against ANGPTL3 mRNA expression.

FIGS. 56A and 56B show inhibition percentages over time of theconjugates of the present disclosure against blood lipid, represented bytotal cholesterol (CHO) and triglyceride (TG) in serum.

FIGS. 57A-57D show inhibition percentages over time of the conjugates ofthe present disclosure against blood lipid, represented by totalcholesterol (CHO) and triglyceride (TG) in serum.

FIGS. 58A and 58B show inhibition percentages over time of theconjugates of the present disclosure against blood lipid, represented bytotal cholesterol (CHO) and triglyceride (TG) in serum; and FIG. 58Cshows the inhibition percentage against ANGPTL3 mRNA expression.

FIG. 59 shows inhibition percentage of the conjugates of the presentdisclosure against APOC3 expression in vitro.

FIG. 60 shows inhibition percentage against APOC3 expression in livertissue on day 14.

FIGS. 61A and 61B show inhibition percentages over time of theconjugates of the present disclosure against blood lipid, represented bytotal cholesterol (CHO) and triglyceride (TG) in serum.

FIGS. 62A and 62B show inhibition percentages over time of theconjugates of the present disclosure against blood lipid, represented bytotal cholesterol (CHO) and triglyceride (TG) in serum.

FIGS. 63A-63D show inhibition percentages over time of the conjugates ofthe present disclosure at different dosages against blood lipid,represented by total cholesterol (CHO) and triglyceride (TG) in serum.

FIGS. 64-65 show the results of the stability tests of the conjugates ofthe present disclosure in vitro.

FIGS. 66-68 show inhibition percentages over time of the conjugates ofthe present disclosure at different dosages against serum surfaceantigen, serum e antigen and HBV DNA.

DETAILED DESCRIPTION OF THE INVENTION

The specific embodiments of the present disclosure are described indetail as below. It should be understood that the specific embodimentsdescribed herein are only for the purpose of illustration andexplanation of the present disclosure and are not intended to limit thepresent disclosure in any respect.

Definitions

In the context of the present disclosure, unless otherwise specified, C,G, U, and A represent the base components of a nucleotide; m representsthat the nucleotide adjacent to the left side of the letter m is a2′-methoxy modified nucleotide; f represents that the nucleotideadjacent to the left side of the letter f is a 2′-fluoro modifiednucleotide; s represents that the two nucleotides adjacent to both sidesof the letter s are linked by a phosphorothioate linkage; P1 representsthat the nucleotide adjacent to the right side of P1 is a 5′-phosphatenucleotide or a 5′-phosphate analog modified nucleotide, especially avinyl phosphate modified nucleotide (expressed as VP in the Examplesbelow), a 5′-phosphate nucleotide (expressed as P in the Examples below)or a 5′-thiophosphate modified nucleotide (expressed as Ps in theExamples below).

In the context of the present disclosure, expressions “complementary”and “reverse complementary” can be interchangeably used, and have awell-known meaning in the art, namely, the bases in one strand arecomplementarily paired with those in the other strand of adouble-stranded nucleic acid molecule. In DNA, a purine base adenine (A)is always paired with a pyrimidine base thymine (T) (or uracil (U) inRNAs); and a purine base guanine (G) is always paired with a pyrimidinebase cytosine (C). Each base pair comprises a purine and a pyrimidine.While adenines in one strand are always paired with thymines (oruracils) in another strand, and guanines are always paired withcytosines, these two strands are considered as being complementary eachother; and the sequence of a strand may be deduced from the sequence ofits complementary strand. Correspondingly, a “mispairing” means that ina double-stranded nucleic acid, the bases at corresponding sites are notpresented in a manner of being complementarily paired.

In the context of the present disclosure, unless otherwise specified,“basically reverse complementary” means that there are no more than 3base mispairings between two nucleotide sequences. “Substantiallyreverse complementary” means that there is no more than 1 basemispairing between two nucleotide sequences. “Completely complementary”means that there is no based mispairing between two nucleotidesequences.

In the context of the present disclosure, when a nucleotide sequence has“nucleotide difference” from another nucleotide sequence, the bases ofthe nucleotides at the same position therebetween are changed. Forexample, if a nucleotide base in the second sequence is A and thenucleotide base at the same position in the first sequence is U, C, G orT, these two nucleotide sequences are considered as having a nucleotidedifference at this position. In some embodiments, if a nucleotide at aposition is replaced with an abasic nucleotide or a nucleotide analogue,it is also considered that there is a nucleotide difference at theposition.

In the context of the present disclosure, particularly in thedescription of the method for preparing the double-strandedoligonucleotide, pharmaceutical composition and/or oligonucleotideconjugate of the present disclosure, unless otherwise specified,“nucleoside monomer” refers to, according to the kind and order of thenucleotides in the double-stranded oligonucleotide, pharmaceuticalcomposition and/or oligonucleotide conjugate to be prepared, unmodifiedor modified nucleoside monomer used in solid phase phosphoramiditesynthesis (unmodified or modified RNA phosphoramidite; RNAphosphoramidites are also referred to as nucleoside phosphoramiditessometimes). Solid phase phosphoramidite synthesis is a well-known methodRNA synthesis to those skilled in the art. Nucleoside monomers used inthe present disclosure can all be commercially available.

As used herein, a dash (“-”) that is not positioned between two lettersor symbols is used to indicate the attachment position of a substituent.For example, —C₁-C₁₀ alkyl-NH₂ is attached through C₁-C₁₀ alkyl.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or condition may or may not occur, and that thedescription includes instances wherein the event or condition may or maynot occur. For example, “optionally substituted alkyl” encompasses both“alkyl” and “substituted alkyl” as defined below. Those skilled in theart would understand, with respect to any group containing one or moresubstituents, that such groups are not intended to introduce anysubstitution or substitution patterns that are sterically impractical,synthetically infeasible and/or inherently unstable.

As used herein, “alkyl” refers to straight chain and branched chainhaving the indicated number of carbon atoms, usually 1 to 20 carbonatoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbonatoms. For example, C₁-C₆ alkyl encompasses both straight and branchedchain alkyl of 1 to 6 carbon atoms. When naming an alkyl residue havinga specific number of carbon atoms, all branched and straight chain formshaving that number of carbon atoms are intended to be encompassed; thus,for example, “butyl” is meant to include n-butyl, sec-butyl, isobutyland t-butyl; “propyl” includes n-propyl and isopropyl. Alkylene is asubset of alkyl, referring to the same residues as alkyl, but having twoattachment positions.

As used herein, “alkenyl” refers to an unsaturated branched orstraight-chain alkyl group having at least one carbon-carbon double bondwhich is obtained by respectively removing one hydrogen molecule fromtwo adjacent carbon atoms of the parent alkyl. The group may be ineither cis or trans configuration of the double bond. Typical alkenylgroups include, but not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl; and the like. In certain embodiments, an alkenylgroup has 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl, referringto the same residues as alkenyl, but having two attachment positions.

As used herein, “alkynyl” refers to an unsaturated branched orstraight-chain alkyl group having at least one carbon-carbon triple bondwhich is obtained by respectively removing two hydrogen molecules fromtwo adjacent carbon atoms of the parent alkyl. Typical alkynyl groupsinclude, but not limited to, ethynyl; propynyls such as prop-1-yn-1-yl,prop-2-yn-1-yl; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl,but-3-yn-1-yl; and the like. In certain embodiments, an alkynyl grouphas 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or2 to 6 carbon atoms. Alkynylene is a subset of alkynyl, referring to thesame residues as alkynyl, but having two attachment positions.

As used herein, “alkoxy” refers to an alkyl group of the indicatednumber of carbon atoms attached through an oxygen bridge, such as, forexample, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy,hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, and the like.Alkoxy groups will usually have 1 to 10, 1 to 8, 1 to 6, or 1 to 4carbon atoms attached through oxygen bridge.

As used herein, “aryl” refers to a radical derived from an aromaticmonocyclic or multicyclic hydrocarbon ring system by removing a hydrogenatom from a ring carbon atom. The aromatic monocyclic or multicyclichydrocarbon ring system contains only hydrogen and carbon, including sixto eighteen carbon atoms, wherein at least one ring in the ring systemis fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system in accordance with the Hückel theory. Aryl groupsinclude, but not limited to, phenyl, fluorenyl, naphthyl and the like.Arylene is a subset of aryl, referring to the same residues as aryl, buthaving two attachment positions.

As used herein, “cycloalkyl” refers to a non-aromatic carbon ring,usually having 3 to 7 ring carbon atoms. The ring may be saturated orhave one or more carbon-carbon double bonds.

Examples of cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl, as well asbridged and caged ring groups such as norbornane.

As used herein, “halo substituent” or “halo” refers to fluoro, chloro,bromo, and iodo, and the term “halogen” includes fluorine, chlorine,bromine, and iodine.

As used herein, “haloalkyl” refers to alkyl as defined above with thespecified number of carbon atoms being substituted with one or morehalogen atoms, up to the maximum allowable number of halogen atoms.Examples of haloalkyl include, but not limited to, trifluoromethyl,difluoromethyl, 2-fluoroethyl, and penta-fluoroethyl.

“Heterocyclyl” refers to a stable 3- to 18-membered non-aromatic ringradical that comprises two to twelve carbon atoms and one to sixheteroatoms selected from nitrogen, oxygen and sulfur. Unless statedotherwise in the description, heterocyclyl is a monocyclic, bicyclic,tricyclic, or tetracyclic ring system, which may include fused orbridged ring systems. The heteroatoms in the heterocyclyl radical may beoptionally oxidized. One or more nitrogen atoms, if present, areoptionally quaternized. The heterocyclyl is partially or fullysaturated. Heterocyclyl may be linked to the rest of the moleculethrough any atom of the ring. Examples of such heterocyclyl include, butnot limited to, dioxanyl, thienyl[1,3]disulfonyl, decahydroisoquinolyl,imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl,2-oxapiperidinyl, 2-oxapyrimidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,trisulfonyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,1-oxa-thiomorpholinyl, and 1,1-dioxa-thiomorpholinyl.

“Heteroaryl” refers to a radical derived from a 3- to 18-memberedaromatic ring radical that comprises two to seventeen carbon atoms andone to six heteroatoms selected from nitrogen, oxygen and sulfur. Asused herein, heteroaryl may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, wherein at least one ring in the ring system isfully unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system in accordance with the Hückel theory.

Heteroaryl includes fused or bridged ring systems. The heteroatom in theheteroaryl radical is optionally oxidized. One or more nitrogen atoms,if present, are optionally quaternized. The heteroaryl is linked to therest of the molecule through any atom of the ring. Examples of suchheteroaryls include, but not limited to, azepinyl, acridinyl,benzimidazolyl, benzindolyl, 1,3-benzodioxazolyl, benzofuranyl,benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxazolyl, benzo[b][1,4]oxazolyl, 1,4-benzodioxazolyl,benzonaphthofuranyl, benzodiazolyl, benzodioxaphenyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl,benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothienyl, furanyl, furanonyl, furo[3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocyclohepta[d]pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, indazolyl,imidazolyl, indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl,indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl,naphthyridinonyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl,oxazolyl, oxalyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl,1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,phthalyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl,pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl,quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6,7,8,9-tetrahydro-5H-cyclohepta [4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl,triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl,thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thienyl.

Various hydroxyl protecting groups can be used in the presentdisclosure. In general, protecting groups render chemicalfunctionalities inert to specific reaction conditions, and can beattached to and removed from such functionalities in a molecule withoutsubstantially damaging the remainder of the molecule. Representativehydroxyl protecting groups are disclosed in Beaucage, et al.,Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts, ProtectiveGroups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, NewYork, 1991, each of which is hereby incorporated by reference in theirentirety. In some embodiments, the protecting group is stable underbasic conditions but can be removed under acidic conditions. In someembodiments, non-exclusive examples of hydroxyl protecting groups usedherein include dimethoxytrityl (DMT), monomethoxytrityl,9-phenylxanthen-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthen-9-yl (Mox).In some embodiments, non-exclusive examples of hydroxyl protectinggroups used herein comprise Tr (trityl), MMTr (4-methoxytrityl), DMTr(4,4′-dimethoxytrityl), and TMTr (4,4′,4″-trimethoxytrityl).

The term “subject”, as used herein, refers to any animal, e.g., mammalor marsupial. Subject of the present disclosure includes, but notlimited to, human, non-human primate (e.g., rhesus or other kinds ofmacaque), mouse, pig, horse, donkey, cow, sheep, rat and any kind ofpoultry.

As used herein, “treatment” or “treating” or “ameliorating” or“improving” are used interchangeably herein. These terms refer to amethod for obtaining advantageous or desired result, including but notlimited to, therapeutic benefit. “Therapeutic benefit” means eradicationor improvement of potential disorder to be treated. Also, therapeuticbenefit is achieved by eradicating or ameliorating one or more ofphysiological symptoms associated with the potential disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the potential disorder.

As used herein, “prevention” and “preventing” are used interchangeably.These terms refer to a method for obtaining advantageous or desiredresult, including but not limited to, prophylactic benefit. Forobtaining “prophylactic benefit”, the conjugate or composition may beadministered to the patient at risk of developing a particular disease,or to the patient reporting one or more physiological symptoms of thedisease, even though the diagnosis of this disease may not have beenmade.

Modified Double-Stranded Oligonucleotide

In one aspect, the present disclosure provides a double-strandedoligonucleotide capable of regulating gene expression.

The double-stranded oligonucleotide of the present disclosure comprisesnucleotides as basic structural units. It is well-known to those skilledin the art that the nucleotide comprises a phosphate group, a ribosegroup and a base. Detailed illustrations relating to such groups areomitted herein.

CN102140458B has disclosed a siRNA that specifically inhibits HBV geneand studied various chemical modification strategies of the siRNA. Thisstudy found that different modification strategies have completelydifferent effects on the parameters of the siRNA, such as stability,biological activity and cytotoxicity. In this study, seven effectivemodification manners were proved. Comparing with unmodified siRNA, thesiRNA obtained by one of the seven modification manners showed increasedstability in blood, while maintaining substantially equal inhibitoryactivity as that of the unmodified siRNA.

The double-stranded oligonucleotide of the present disclosure comprisesa sense strand and an antisense strand, each nucleotide in the sensestrand and antisense strand being a modified nucleotide, wherein thesense strand comprises a nucleotide sequence 1, and the antisense strandcomprises a nucleotide sequence 2; the nucleotide sequence 1 and thenucleotide sequence 2 are both 19 nucleotides in length and are at leastpartly reverse complementary to form a double-stranded complementaryregion; the nucleotide sequence 2 is at least partly reversecomplementary to a first nucleotide sequence segment, which refers to asegment of nucleotide sequence in the target mRNA; in the direction from5′ terminal to 3′ terminal, the nucleotides at positions 7, 8 and 9 ofthe nucleotide sequence 1 are fluoro modified nucleotides, and each ofthe nucleotides at the other positions in the nucleotide sequence 1 isindependently a non-fluoro modified nucleotide; the first nucleotide at5′ terminal of the nucleotide sequence 2 is the first nucleotide at 5′terminal of the antisense strand, the nucleotides at positions 2, 6, 14and 16 of the nucleotide sequence 2 are fluoro modified nucleotides, andeach of the nucleotides at the other positions in the nucleotidesequence 2 is independently a non-fluoro modified nucleotide. In thecontext of the present disclosure, a “fluoro modified nucleotide” refersto a nucleotide formed by substituting the 2′-hydroxy of the ribosegroup with a fluorine atom; and a “non-fluoro modified nucleotide”refers to a nucleotide formed by substituting the 2′-hydroxy of theribose group with a non-fluoro group, or a nucleotide analogue.

A “nucleotide analogue” refers to a group that can replace a nucleotidein a nucleic acid, while structurally differs from an adenineribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, auracil ribonucleotide or thymine deoxyribonucleotide, such as anisonucleotide, a bridged nucleic acid (BNA) nucleotide or an acyclicnucleotide.

In some embodiments, the nucleotide sequence 2 is basically reversecomplementary, substantially reverse complementary, or completelyreverse complementary to the first nucleotide sequence segment.

In some embodiments, in the direction from 5′ terminal to 3′ terminal,at least the nucleotides at positions 2-19 of the nucleotide sequence 2are complementary to the first nucleotide sequence segment. In somespecific embodiments, in the direction from 5′ terminal to 3′ terminal,the nucleotide at position 1 of the nucleotide sequence 2 is A or U.

In some embodiments, the nucleotide sequence 1 is basically reversecomplementary, substantially reverse complementary, or completelyreverse complementary to the nucleotide sequence 2.

In some embodiments, the sense strand further comprises nucleotidesequence 3, and the antisense strand further comprises nucleotidesequence 4; each nucleotide in the nucleotide sequence 3 and thenucleotide sequence 4 is independently a non-fluoro modified nucleotide;the nucleotide sequence 3 and the nucleotide sequence 4 are respectively1-4 nucleotides in length; the nucleotide sequence 3 and the nucleotidesequence 4 have equal length and are substantially reverse complementaryor complete reverse complementary to each other; the nucleotide sequence3 is linked to 5′ terminal of the nucleotide sequence 1; and thenucleotide sequence 4 is linked to the 3′ terminal of the nucleotidesequence 2; the nucleotide sequence 4 is substantially reversecomplementary, or completely reverse complementary to a secondnucleotide sequence segment, which refers to a nucleotide sequence thatis adjacent to the first nucleotide sequence segment in the target mRNAand has the same length as the nucleotide sequence 4.

In some embodiments, the nucleotide sequence 3 is complete complementaryto the nucleotide sequence 4, the nucleotide sequence 3 and thenucleotide sequence 4 are both 1 nucleotide in length, and thenucleotide sequence 4 is completely reverse complementary to the secondnucleotide sequence segment; or the nucleotide sequence 3 is completecomplementary to the nucleotide sequence 4, the nucleotide sequence 3and the nucleotide sequence 4 are both 2 nucleotides in length, and thenucleotide sequence 4 is completely reverse complementary to the secondnucleotide sequence segment; or the nucleotide sequence 3 is completecomplementary to the nucleotide sequence 4, the nucleotide sequence 3and the nucleotide sequence 4 are both 3 nucleotides in length, and thenucleotide sequence 4 is completely reverse complementary to the secondnucleotide sequence segment; or the nucleotide sequence 3 is completecomplementary to the nucleotide sequence 4, the nucleotide sequence 3and the nucleotide sequence 4 are both 4 nucleotides in length, and thenucleotide sequence 4 is completely reverse complementary to the secondnucleotide sequence segment.

The nucleotide sequence 3 is completely reverse complementary to thenucleotide sequence 4, the nucleotide sequence 4 is completely reversecomplementary to the second nucleotide sequence segment, and once therelevant nucleotide sequence of the target mRNA is determined, thenucleotide sequence 3 and nucleotide sequence 4 are also determined.

Therefore, the sense strand or the antisense strand may independently be19-23 nucleotides in length.

In some embodiments, the double-stranded oligonucleotide also comprisesa nucleotide sequence 5; each nucleotide in the nucleotide sequence 5 isindependently a non-fluoro modified nucleotide; the nucleotide sequence5 is 1-3 nucleotides in length and is linked to 3′ terminal of theantisense strand, thereby forming a 3′ overhang of the antisense strand.

As such, the length ratio of the sense strand to the antisense strand inthe double-stranded oligonucleotide of the present disclosure may be19/19, 19/20, 19/21, 19/22, 20/20, 20/21, 20/22, 20/23, 21/21, 21/22,21/23, 21/24, 22/22, 22/23, 22/24, 22/25, 23/23, 23/24, 23/25 or 23/26.

In some embodiments, the nucleotide sequence 5 is 2 nucleotides inlength. Moreover, the nucleotide sequence 5 is 2 consecutive thymidinedeoxynucleotides, or 2 consecutive uridine nucleotides in the directionfrom 5′ terminal to 3′ terminal, or completely reverse complementary toa third nucleotide sequence segment, which refers to a nucleotidesequence that is adjacent to the first or second nucleotide sequencesegment in the target mRNA and has the same length as the nucleotidesequence 5.

Therefore, in some embodiments, the length ratio of the sense strand tothe antisense strand in the double-stranded oligonucleotide of thepresent disclosure is 19/21 or 21/23. Here, the double-strandedoligonucleotide of the present disclosure exhibits better silencingactivity against target mRNA.

In the context of the present disclosure, a “fluoro modified nucleotide”refers to a nucleotide formed by substituting the 2′-hydroxy of theribose group with a fluorine atom, as shown by Formula (101), wherein“Base” represents a base selected from C, G, A, and U.

A “non-fluoro modified nucleotide” refers to a nucleotide formed bysubstituting the 2′-hydroxy of the ribose group with a non-fluoro group,or a nucleotide analogue. In some embodiments, each non-fluoro modifiednucleotide is independently selected from the group consisting of anucleotide formed by substituting the 2′-hydroxy of the ribose groupthereof with a non-fluoro group, and a nucleotide analogue.

A nucleotide formed by substituting the 2′-hydroxy of the ribose groupwith a non-fluoro group is well-known to those skilled in the art, suchas 2′-alkoxy modified nucleotide, 2′-substituted alkoxy modifiednucleotide, 2′-alkyl modified nucleotide, 2′-substituted alkyl modifiednucleotide, 2′-amino modified nucleotide, 2′-substituted amino modifiednucleotide or 2′-deoxy nucleotide.

In some embodiments, the 2′-alkoxy modified nucleotide is a methoxymodified nucleotide (2′-OMe), as shown by Formula (102). In someembodiments, the 2′-substituted alkoxy modified nucleotide is a2′-O-methoxyethyl modified nucleotide (2′-MOE), as shown by Formula(103). In some embodiments, the 2′-amino modified nucleotide (2′-NH₂) isas shown by Formula (104). In some embodiments, the 2′-deoxy nucleotide(DNA) is as shown by Formula (105).

A “nucleotide analogue” refers to a group that can replace a nucleotidein the nucleic acid, while structurally differs from an adenineribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, auracil ribonucleotide or thymine deoxyribonucleotide. In someembodiments, the nucleotide analogue may be an isonucleotide, a bridgednucleic acid (BNA) nucleotide or an acyclic nucleotide.

A BNA is a nucleotide that is constrained or is not accessible. BNA cancontain a 5-, 6-membered or even a 7-membered ring bridged structurewith a “fixed” C3′-endo sugar puckering. The bridge is typicallyincorporated at the 2′- and 4′-position of the ribose to afford a 2′,4′-BNA nucleotide, such as LNA, ENA and cET BNA, which are as shown byFormulae (106), (107) and (108), respectively:

An acyclic nucleotide is a “ring-opened” nucleotide in which the sugarring is opened, such as an unlocked nucleic acid (UNA) and a glycerolnucleic acid (GNA), which are as shown by Formulae (109) and (110),respectively:

wherein R is H, OH or alkoxy (O-alkyl).

An isonucleotide is a nucleotide in which the position of the base onthe ribose ring is changed, such as a compound in which the base istransposed from position-1′ to position-2′ or -3′ on the ribose ring, asshown by Formula (111) or (112):

wherein Base represents a base, such as A, U, G, C or T; R is selectedfrom the group consisting of H, OH, F, and a non-fluoro group describedabove.

In some embodiments, a nucleotide analogue is selected from the groupconsisting of an isonucleotide, LNA, ENA, cET, UNA, and GNA. In someembodiments, each non-fluoro modified nucleotide is a methoxy modifiednucleotide. In the context of the present disclosure, the methoxymodified nucleotide refers to a nucleotide formed by substituting the2′-hydroxy of the ribose group with a methoxy group.

In the context of the present disclosure, a “fluoro modifiednucleotide”, a “2′-fluoro modified nucleotide”, a “nucleotide in which2′-hydroxy of the ribose group is substituted with fluoro” and a“2′-fluororibosyl” have the same meaning, referring to the nucleotideformed by substituting the 2′-hydroxy of the ribose group with fluoro,having a structure as shown by Formula (101). A “methoxy modifiednucleotide”, a “2′-methoxy modified nucleotide”, a “nucleotide in which2′-hydroxy of a ribose group is substituted with methoxy” and a“2′-methoxyribosyl” have the same meaning, referring to the nucleotideformed by substituting the 2′-hydroxy of the ribose group with methoxy,having a structure as shown by Formula (102).

In some embodiments, the double-stranded oligonucleotide of the presentdisclosure can resist ribonuclease cleavage in blood, thereby enhancingthe stability of the nucleic acid in blood and allowing the nucleic acidto have stronger resistance against nuclease hydrolysis, whilemaintaining higher activity for regulating the target gene.

In some embodiments, the double-stranded oligonucleotides of the presentdisclosure realize high balance between the stability in serum and theefficiency of regulating gene expression in animal experiments; and somealso have the advantages such as simpler structure and lower cost. Thefollowing are some examples.

In the direction from 5′ terminal to 3′ terminal, the nucleotides atpositions 7, 8 and 9 of the nucleotide sequence 1 in the sense strandare fluoro modified nucleotides, and the nucleotides at the otherpositions in the sense strand are methoxy modified nucleotides; and thenucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence 2in the antisense strand are fluoro modified nucleotides, and thenucleotides at the other positions in the antisense strand are methoxymodified nucleotides.

In some embodiments, the double-stranded oligonucleotides of the presentdisclosure further comprise additional modified nucleotide(s) whichwould not result in significant impairment or loss of the function ofthe double-stranded oligonucleotides for regulating the expression ofthe target gene.

Currently, there are many means in the art that can be used to modifydouble-stranded oligonucleotides, including the ribose groupmodification mentioned above, backbone modification (such as phosphategroup modification), base modification, and the like (see, for example,Watts, J. K., G. F. Deleavey and M. J. Damha, Chemically Modified siRNA:tools and applications. Drug Discov Today, 2008.13(19-20): p. 842-55,which is incorporated herein by reference in its entirety).

In some embodiments, at least one phosphate group in thephosphate-ribose backbone of at least one single strand of the sensestrand and the antisense strand is a phosphate group with modifiedgroup(s). The phosphate group with modified group(s) is aphosphorothioate group formed by substituting at least one of oxygenatoms in a phosphodiester bond in the phosphate groups with a sulfuratom, such as the phosphorothioate structure as shown by Formula (121)below, that is, substituting a non-bridging oxygen atom in aphosphodiester bond with a sulfur atom such that the phosphodiester bondis changed to a phosphorothioate diester bond; in other words, thelinkage between two nucleotides is a phosphorothioate linkage. Thismodification could stabilize the structure of the double-strandedoligonucleotide, while maintain high specificity and high affinity ofbase pairing.

In some embodiments, in the double-stranded oligonucleotides, aphosphorothioate linkage exists in at least one of the followingpositions: the position between the first and the second nucleotides ateither terminal of the sense or antisense strand, the position betweenthe second and the third nucleotides at either terminal of the sense orantisense strand, or any combination thereof. In some embodiments, aphosphorothioate linkage exists at all the above positions except for 5′terminal of the sense strand. In some embodiments, a phosphorothioatelinkage exists at all the above positions except for 3′ terminal of thesense strand. In some embodiments, a phosphorothioate linkage exists inat least one of the following positions:

the position between the first and second nucleotides at 5′ terminal ofthe sense strand;the position between the second and third nucleotides at 5′ terminal ofthe sense strand;the position between the first and second nucleotides at 3′ terminal ofthe sense strand;the position between the second and third nucleotides at 3′ terminal ofthe sense strand;the position between the first and second nucleotides at 5′ terminal ofthe antisense strand;the position between the second and third nucleotides at 5′ terminal ofthe antisense strand;the position between the first and second nucleotides at 3′ terminal ofthe antisense strand; andthe position between the second and third nucleotides at 3′ terminal ofthe antisense strand.

In some embodiments, the nucleotide at 5′-terminal of the antisensestrand of the double-stranded oligonucleotide molecule is a 5′-phosphatenucleotide or a 5′-phosphate analogue modified nucleotide.

In some embodiments, the 5′-phosphate nucleotide has the structure asshown by Formula (122):

Meanwhile, common 5′-phosphate analogue modified nucleotides are wellknown to those skilled in the art, for example, the followingnucleotides shown by Formulae (123)-(126) as disclosed in AnastasiaKhvorova and Jonathan K. Watts, The chemical evolution ofoligonucleotide therapies of clinical utility. Nature Biotechnology,2017, 35(3): 238-48:

wherein R represents a group selected from the group consisting of H,OH, F, and methoxy; and “Base” represents a base selected from A, U, C,G, and T.

In some embodiments, the 5′-phosphate analogue modified nucleotide is anucleotide containing an E-vinylphosphate (E-VP) as shown by Formula(123), or a nucleotide containing phosphorothioate as shown by Formula(125).

The modification strategies of the present disclosure are suitable forvarious double-stranded oligonucleotides for regulating gene expression.In some embodiments, they may be double-stranded oligonucleotides (suchas siRNA) that inhibit or down-regulate gene expression; in someembodiments, they may be double-stranded oligonucleotides (such assaRNA) that activate or up-regulate gene expression.

By using the modification strategies of the present disclosure, theresultant double-stranded oligonucleotides have unexpectedly enhancedstability in blood, increased stability in lysosome, reduced off-targeteffect, and/or increased activity, without significantly reducedactivity for regulating the expression of the target gene, while showingexcellent inhibitory effect in vivo.

The modified double-stranded oligonucleotide, pharmaceutical compositionand conjugate of the present disclosure can be used for regulatingvarious abnormal gene expressions and for treating various pathologicalconditions or diseases caused by abnormal gene expression. These genesmay be various endogenous genes in human or animal bodies, or the genesof pathogens reproduced in human or animal bodies. Double-strandedoligonucleotides with specific nucleotide sequences and saidmodification strategies may be designed and prepared according to thetarget mRNA of interest.

According to some embodiments of the present disclosure, thedouble-stranded oligonucleotides of the present disclosure could, forexample, be the following siRNAs:

the nucleotide sequence 1 is a sequence shown by SEQ ID NO: 1, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 2; or

the nucleotide sequence 1 is a sequence shown by SEQ ID NO: 3, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 4; or

the nucleotide sequence 1 is a sequence shown by SEQ ID NO: 5, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 6; or

the nucleotide sequence 1 is a sequence shown by SEQ ID NO: 7, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 8; or

the nucleotide sequence 1 is a sequence shown by SEQ ID NO: 9, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 10; or

the nucleotide sequence 1 is a sequence shown by SEQ ID NO: 11, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 12; or

the nucleotide sequence 1 is a sequence shown by SEQ ID NO: 13, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 14;

(SEQ ID NO: 1) 5′- CmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm -3′(SEQ ID NO: 2) 5′- UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGm -3′(SEQ ID NO: 3) 5′- UmGmCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm -3′(SEQ ID NO: 4) 5′- UmAfGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAm -3′(SEQ ID NO: 5) 5′- UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm -3′(SEQ ID NO: 6) 5′- UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAm -3′(SEQ ID NO: 7) 5′- CmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm -3′(SEQ ID NO: 8) 5′- UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGm -3′(SEQ ID NO: 9) 5′- GmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm -3′(SEQ ID NO: 10) 5′- UmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCm -3′(SEQ ID NO: 11) 5′- CmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm -3′(SEQ ID NO: 12) 5′- UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGm -3′(SEQ ID NO: 13) 5′- CmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm -3′(SEQ ID NO: 14) 5′- UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGm -3′wherein C, G, U, and A represent the base components of the nucleotides;m represents that the nucleotide adjacent to the left side of the letterm is a 2′-methoxy modified nucleotide; f represents that the nucleotideadjacent to the left side of the letter f is a 2′-fluoro modifiednucleotide.

According to some embodiments of the present disclosure, thedouble-stranded oligonucleotides of the present disclosure may be, forexample, the siRNAs as shown in Tables 1A-1F:

Table 1 siRNA in some embodiments

TABLE 1A SEQ ID No Sequence Direction: 5′-3′ NO siHBa1M1 SCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm 15 ASUmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmUm 16 siHBa2M1 SGmAmCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm 17 ASUmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmGmGm 18 siHBa1M1S SCmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm 19 ASUmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm 20 siHBa2M1S SGmsAmsCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm 21 ASUmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmsGmsGm 22 siHBa1M1P1 SCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm 23 ASP1-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmUm 24 siHBa2M1P1 SGmAmCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm 25 ASP1-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmGmGm 26 SiHBa1M1SP1 SCmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm 27 ASP1-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm 28 siHBa2M1SP1 SGmsAmsCmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm 29 ASP1-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmsGmsGm 30

TABLE 1B SEQ ID No Sequence Direction: 5′-3′ NO SiHBb1 SUGCUAUGCCUCAUCUUCUA 31 AS UAGAAGAUGAGGCAUAGCAGC 32 siHBb1M1 SUmGmCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm 33 ASUmAfGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAmGmCm 34 siHBb2M1 SUmGmCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm 35 ASUmAfGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAmUmUm 36 siHBb1M1S SUmsGmsCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm 37 ASUmsAfsGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAmsGmsCm 38 siHBb2M1S SUmsGmsCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm 39 ASUmsAfsGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAmsUmsUm 40 siHBb1M1P1 SUmGmCmUmAmUmGfCfCfUmCmamUmCmUmUmCmUmAm 41 ASP1-UmAfGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAmGmCm 42 siHBb2M1P1 SUmGmCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm 43 ASP1-UmAfGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAmUmUm 44 siHBb1M1SP1 SUmsGmsCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm 45 ASP1-UmsAfsGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAmsGmsCm 46 siHBb2M1SP1 SUmsGmsCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm 47 ASP1-UmsAfsGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAmsUmsUm 48

TABLE 1C SEQ ID No Sequence Direction: 5′-3′ NO siHBc1M1 SUmCmUmGmUmGmfCfUfUmCmUmCmAmUmCmUmGmAm 49 ASUmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmCmGm 50 siHBc1M1S SUmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm 51 ASUmsCfsAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmsCmsGm 52 SiHBc1M1P1 SUmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm 53 ASP1-UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmCmGm 54 siHBc1M1SP1 SUmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm 55 ASP1-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAmsCmsGm 56

TABLE 1D SEQ ID No Sequence Direction: 5′-3′ NO siHBd1M1 SCmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm 57 ASUmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmGmUm 58 siHBd1M1S SCmsGmsUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm 59 ASUmsUfsGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmsGmsUm 60 siHBd1M1P1 SCmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm 61 ASP1-UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmGmUm 62 siHBd1M1SP1 SCmsGmsUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm 63 ASP1-UmsUfsGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGmsGmsUm 64

TABLE 1E SEQ ID No Sequence Direction: 5′-3′ NO siAN1M3 SCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm 65 ASUmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUm 66 siAN2M3 SAmGmCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm 67 ASUmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmUmGm 68 siAN1M3S SCmsCmsAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm 69 ASUmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsCmsUm 70 siAN2M3S SAmsGmsCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm 71 ASUmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmsUmsGm 72 siAN1M3P1 SCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmcmUmAm 73 ASP1-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUm 74 siAN2M3P1 SAmGmCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm 75 ASP1-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmUmGm 76 siAN1M3SP1 SCmsCmsAmAmGmAmGfCfAfCmCmAmAmgmAmAmCmUmAm 77 ASP1-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsCmsUm 78 siAN2M3SP1 SAmsGmsCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm 79 ASP1-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmsUmsGm 80

TABLE 1F SEQ ID No Sequence Direction: 5′-3′ NO siAP1M2 SCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm 81 ASUmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGm 82 siAP2M2 SCmCmCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm 83 ASUmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGmAmGm 84 siAP1M2S SCmsAmsAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm 85 ASUmsUfsCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmsGmsGm 86 siAP2M2S SCmsCmsCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm 87 ASUmsUfsCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGmsAmsGm 88 siAP1M2P1 SCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm 89 ASP1-UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGm 90 siAP2M2P1 SCmCmCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm 91 ASP1-UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGmAmGm 92 siAP1M2SP1 SCmsAmsAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm 93 ASP1-UmsUfsCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmsGmsGm 94 siAP2M2SP1 SCmsCmsCmamamUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm 95 ASP1-UmsUfsCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGmsAmsGm 96

TABLE 1G SEQ ID No Sequence Direction: 5′-3′ NO siHBc1M1 SGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmUm  97 ASAmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmUmUm  98 siHBc2M1 SGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmUm  99 ASAmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmCmAm 100 siHBc3M1 SGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 101 ASUmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmUmUm 102 siHBc4M1 SGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 103 ASUmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmCmAm 104 siHBc5M1 SUmGmGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 105 ASUmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmCmAmUmUm 106 siHBc1M1S SGmsAmsAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmUm 107 ASAmsAfsUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmsUmsUm 108 siHBc2M1S SGmsAmsAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmUm 109 ASAmsAfsUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmsCmsAm 110 siHBc3M1S SGmsAmsAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 111 ASUmsAfsUmUmCmGfUmUmGmAmCmAmCmAfCmUfUmUmCmsUmsUm 112 siHBc4M1S SGmsAmsAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 113 ASUmsAfsUmUmCmGfUmUmGmAmCmAmCmAfCmUfUmUmCmsCmsAm 114 siHBc5M1S SUmsGmsGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 115 ASUmsAfsUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmCmAmsUmsUm 116 siHBc1M1P SGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmUm 117 ASP1-AmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmUmUm 118 siHBc2M1P SGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmUm 119 ASP1-AmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmCmAm 120 siHBc3M1P SGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 121 ASP1-UmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmUmUm 122 siHBc4M1P SGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 123 ASP1-UmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmCmAm 124 siHBc5M1P SUmGmGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 125 ASP1-UmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmCmAmUmUm 126 siHBc1M1SP SGmsAmsAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmUm 127 ASP1-AmsAfsUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmsUmsUm 128 siHBc2M1SP SGmsAmsAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmUm 129 ASP1-AmsAfsUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmsCmsAm 130 siHBc3M1SP SGmsAmsAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 131 ASP1-UmsAfsUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmsUmsUm 132 siHBc4M1SP SGmsAmsAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 133 ASP1-UmsAfsUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmsCmsAm 134 siHBc5M1SP SUmsGmsGmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm 135 ASP1-UmsAfsUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCmCmAmsUmsUm 136 *S: SenseStrand; AS: Antisense Strand

In the above tables, C, G, U, and A represent the base components of thenucleotides; m represents that the nucleotide adjacent to the left sideof the letter in is a 2′-methoxy modified nucleotide; f represents thatthe nucleotide adjacent to the left side of the letter f is a 2′-fluoromodified nucleotide; s represents that the two nucleotides adjacent toboth sides of the letter s are linked by a phosphorothioate linkage; P1represents that the nucleotide adjacent to the right side of P1 is a5′-phosphate nucleotide or a 5′-phosphate analogue modified nucleotide,and in some embodiments, is a vinyl phosphate modified nucleotide(expressed as VP in the Examples below), a 5′-phosphate modifiednucleotide (expressed as P in the Examples below) or a phosphorothioatemodified nucleotide (expressed as Ps in the Examples below).

Those skilled in the art clearly know that the double-strandedoligonucleotides of the present disclosure can be obtained byconventional methods in the art for preparing double-strandedoligonucleotides, e.g., solid phase synthesis and liquid phasesynthesis. Solid phase synthesis already has commercial customizationservice. A modified nucleotide can be introduced into thedouble-stranded oligonucleotide of the present disclosure by using anucleotide monomer having a corresponding modification, wherein themethods for preparing a nucleotide monomer having the correspondingmodification and the methods for introducing a modified nucleotide intoa double-stranded oligonucleotide are also well-known to those skilledin the art.

The modified double-stranded oligonucleotides of the present disclosurecan be used alone, or as a pharmaceutical composition by combining witha pharmaceutically acceptable carrier, as a conjugate by binding to aconjugating molecule, or in other forms. An effective amount of thedouble-stranded oligonucleotide, pharmaceutical composition or conjugateare contacted with a cell to regulate expression of the target gene, orthe double-stranded oligonucleotide, pharmaceutical composition orconjugate is administered to a subject to regulate expression of thetarget gene, thereby achieving the purpose of treating a pathologicalcondition or disease caused by abnormal expression of the target gene.

By forming a pharmaceutical composition with a suitable carrier orforming a conjugate with a suitable conjugating molecule, thedouble-stranded oligonucleotide of the present disclosure could haveimproved stability in blood and increased targeting property, and solvedits in vivo delivery problem, etc. For double-stranded oligonucleotides,carriers or conjugating molecules that can confer or improve targetingproperty will be very advantageous, since they can significantlyincrease the efficiency of regulating the expression of the target geneand decrease potential side effects of the double-strandedoligonucleotide. Furthermore, after introducing a targeting carrier orconjugating molecule, the double-stranded oligonucleotide still needs toexet function at the target site, i.e., the encapsulation/conjugation bythe carrier or conjugating molecule cannot affect the activity of thedouble-stranded oligonucleotide (for example, in the case where thedouble-stranded oligonucleotide is siRNA, cannot affect the loading ofthe siRNA into the RNAi machinery in cells, i.e., the RISC complex). Inaddition, such targeting carriers or conjugating molecules also need tohave good biocompatibility and minimal toxicity.

The pharmaceutical composition can be systemically distributed atvarious sites of the body or targetedly enriched at a specific site ofthe body. The conjugate generally has targeting property, and theconjugating molecule can be adaptively changed according to theexpression profile of the target gene in a human or animal body, so asto achieve the purpose of delivering the double-stranded oligonucleotideto the relevant site. For example, the conjugating molecule may be aconjugating molecule targeting liver, lung, kidney, or cancer cells.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition, comprising the modified double-stranded oligonucleotideabove and a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier may be any suitable carrier.

In some embodiments, the present disclosure provides an oligonucleotideconjugate, comprising the modified double-stranded oligonucleotide aboveand a ligand conjugated to the double-stranded oligonucleotide.According to the expression profile of the target gene, differentconjugating molecules may be adopted to deliver the double-strandedoligonucleotide to different organs or cells. The conjugating moleculesbelow are suitable for delivering the double-stranded oligonucleotide toliver, thereby regulating the expression of an endogenous gene expressedin the target liver or the expression of a gene of a pathogen reproducedin the liver, so as to achieve the purpose of treating a pathologicalcondition or disease caused by abnormal expression of the endogenousgene expressed in the liver or the gene of the pathogen reproduced inthe liver.

In some embodiments, the present disclosure provides use of the abovedouble-stranded oligonucleotide above, the above pharmaceuticalcomposition comprising the double-stranded oligonucleotide, or the aboveoligonucleotide conjugate in the manufacture of a medicament fortreating and/or preventing a pathological condition or disease caused bygene overexpression.

In some embodiments, the present disclosure provides a method fortreating a pathological condition or disease caused by abnormal geneexpression, comprising administering an effective amount of the abovedouble-stranded oligonucleotide, the above pharmaceutical composition orthe above oligonucleotide conjugate to a subject.

In some embodiments, the present disclosure provides a method forregulating the expression of a gene, comprising contacting an effectiveamount of the above double-stranded oligonucleotide, the abovepharmaceutical composition or the above oligonucleotide conjugate with acell expressing said gene. In some embodiments, the abnormal expressionis overexpression; correspondingly, the regulation refers to inhibitingthe overexpression.

In some embodiments, the above double-stranded oligonucleotide, theabove pharmaceutical composition or the above oligonucleotide conjugateexhibits unexpected stability and activity in the regulation of a geneexpressed in liver or in the treatment of a pathological condition ordisease caused by abnormal expression of a gene in a liver cell. Thegene expressed in liver includes, but not limited to, ApoB, ApoC,ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, and HCV. Insome embodiments, the specific gene is selected from the gene ofhepatitis B virus, the gene of angiopoietin-like protein 3, and the geneof apolipoprotein C3. Correspondingly, the diseases are selected fromchronic liver diseases, hepatitis, hepatic fibrosis, liver proliferativediseases, and dyslipidemia. In some embodiments, the dyslipidemia ishypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

In some embodiments, the above double-stranded oligonucleotide, theabove pharmaceutical composition or the above oligonucleotide conjugatemay also be used to treat other liver diseases, including diseasescharacterized by undesired cell proliferation, blood diseases, metabolicdiseases, and diseases characterized by inflammation. Liverproliferative diseases may be benign or malignant diseases, such ascancer, hepatocellular carcinoma (HCC), hepatic metastasis orhepatoblastoma. Liver hematology or inflammatory diseases may bediseases involving coagulation factors, complement-mediated inflammationor fibrosis. Liver metabolic diseases include dyslipidemia and irregularglucose regulation.

The present disclosure provides a kit comprising the abovedouble-stranded oligonucleotide, the above pharmaceutical composition orthe above oligonucleotide conjugate.

The following description of pharmaceutical compositions andoligonucleotide conjugates is based on the aforementioneddouble-stranded oligonucleotides suitable for regulating geneexpression. However, the description regarding pharmaceuticallyacceptable carriers and ligands in drug conjugates are also applicableto systemic administration of said modified double-strandedoligonucleotides and delivery of said double-stranded oligonucleotidesto the target organs or tissues, particularly liver, for regulating theexpression of an endogenous gene expressed in the target organ or tissueor the expression of a gene of a pathogen reproduced in the target organor tissue.

Pharmaceutical Composition

In one aspect, the present disclosure provides a pharmaceuticalcomposition, comprising the above double-stranded oligonucleotide as anactive ingredient, and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be a conventional carrierused in the field of double-stranded oligonucleotide administration, forexample, but not limited to, one or more of magnetic nanoparticles (suchas Fe₃O₄ and Fe₂O₃-based nanoparticle), carbon nanotubes, mesoporoussilicon, calcium phosphate nanoparticles, polyethylenimine (PEI),polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),poly(D&L-lactic/glycolic acid) copolymer (PLGA), poly(2-aminoethylethylene phosphate) (PPEEA), poly(2-dimethylaminoethyl methacrylate)(PDMAEMA), and derivatives thereof.

According to some embodiments, in the pharmaceutical composition of thepresent invention, there are no special requirements for the contents ofthe double-stranded oligonucleotide and the pharmaceutically acceptablecarrier. In some embodiments, the ratio of the double-strandedoligonucleotide to the pharmaceutically acceptable carrier is 1:(1-500)by weight; in some embodiments, the ratio is 1:(1-50) by weight.

In some embodiments, the pharmaceutical composition of the presentinvention may also contain other pharmaceutically acceptable excipients,which may be one or more of various conventional formulations orcompounds in the art. For example, said other pharmaceuticallyacceptable excipients may comprise at least one of a pH buffer, aprotective agent and an osmotic pressure regulator.

The pH buffer may be a tris(hydroxymethyl) aminomethane hydrochloridebuffer solution with a pH of 7.5-8.5, and/or a phosphate buffer solutionwith a pH of 5.5-8.5, preferably a phosphate buffer solution with a pHof 5.5-8.5.

The protective agent may be at least one of inositol, sorbitol, sucrose,trehalose, mannose, maltose, lactose, and glucose. The content of theprotective agent may be from 0.01 wt % to 30 wt % on the basis of thetotal weight of the pharmaceutical composition.

The osmotic pressure regulator may be sodium chloride and/or potassiumchloride. The content of the osmotic pressure regulator allows theosmotic pressure of the pharmaceutical composition to be 200-700milliosmol/kg. Depending on the desired osmotic pressure, those skilledin the art can readily determine the content of the osmotic pressureregulator.

In some embodiments, the pharmaceutical composition may be a liquidformulation, for example, an injection solution; or a lyophilized powderfor injection, which is mixed with a liquid excipient to form a liquidformulation upon administration. The liquid formulation may beadministered by, but not limited to, subcutaneous, intramuscular orintravenous injection routes, and also may be administered to, but notlimited to, lung by spray, or other organs (such as liver) via lung byspray. In some specific embodiments, the pharmaceutical composition isadministered by intravenous injection.

In some embodiments, the pharmaceutical composition may be in the formof a liposome formulation. In some embodiments, the pharmaceuticallyacceptable carrier used in the liposome formulation comprises anamine-containing transfection compound (hereinafter also referred to asan organic amine), a helper lipid and/or a PEGylated lipid. Therein, theorganic amine, the helper lipid and the PEGylated lipid may berespectively selected from one or more of the amine-containingtransfection compounds or the pharmaceutically acceptable salts orderivatives thereof, the helper lipids and the PEGylated lipids asdescribed in CN103380113A, which is incorporated herein by reference inits entirety.

In some embodiments, the organic amine may be a compound as shown byFormula (201) as described in CN103380113A or a pharmaceuticallyacceptable salt thereof:

wherein:

X₁₀₁ and X₁₀₂ independently of one another are selected from O, S, N-Aand C-A, wherein A is hydrogen or a C₁-C₂₀ hydrocarbon chain;

Y and Z independently of one another are selected from C═O, C═S, S═O,CH—OH and SO₂; R₁₀₁, R₁₀₂, R₁₀₃, R₁₀₄, R₁₀₅, R₁₀₆ and R₁₀₇ independentlyof one another are selected from hydrogen; a cyclic or an acyclic,substituted or unsubstituted, branched or linear aliphatic group; acyclic or an acyclic, substituted or unsubstituted, branched or linearheteroaliphatic group; a substituted or unsubstituted, branched orlinear acyl group; a substituted or unsubstituted, branched or lineararyl group, or a substituted or unsubstituted, branched or linearheteroaryl group;

x is an integer of 1-10;

n is an integer of 1-3, m is an integer of 0-20, p is 0 or 1; wherein ifm and p are both 0, then R₁₀₂ is hydrogen, and

if at least one of n or m has is 2, then R₁₀₃ and nitrogen in Formula(201) form a structure as shown by Formula (202) or (203):

wherein g, e and f independently of one another are an integer of 1-6,“HCC” represents a hydrocarbon chain, and each *N represents a nitrogenatom shown in Formula (201).

In some embodiments, R₁₀₃ is a polyamine. In other embodiments, R₁₀₃ isa ketal. In some embodiments, R₁₀₁ and R₁₀₂ in the Formula (201)independently of one another are any of substituted or unsubstituted,branched or linear alkyl or alkenyl groups which have 3-20 carbon atoms(such as 8-18 carbon atoms) and 0-4 double bonds (such as 0-2 doublebonds).

In some embodiments, if n and m independently of one another are 1-3,R₁₀₃ represents any of the following Formulae (204)-(213):

wherein each “HCC” represents a hydrocarbon chain, and each * representsa potential attachment point of R₁₀₃ to the nitrogen atom in Formula(201), where each H at any * position can be replaced to realize theattachment to the nitrogen atom in Formula (201).

The compound as shown by Formula (201) may be prepared as described inCN103380113A.

In some specific embodiments, the organic amine may be an organic amineas shown by Formula (214) and/or an organic amine as shown by Formula(215):

The helper lipid is cholesterol, cholesterol analogue and/or cholesterolderivatives.

The PEGylated lipid is1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethyleneglycol)]-2000.

In some embodiments, the molar ratio among the organic amine, the helperlipid, and the PEGylated lipid in the pharmaceutical composition is(19.7-80):(19.7-80):(0.3-50); for example, the molar ratio may be(50-70):(20-40):(3-20).

In some embodiments, the pharmaceutical composition particles formed bythe double-stranded oligonucleotide of the present disclosure and theabove amine-containing transfection agents have an average diameter fromabout 30 nm to about 200 nm, typically from about 40 nm to about 135 nm,and more typically, the average diameter of the liposome particles isfrom about 50 nm to about 120 nm, from about 50 nm to about 100 nm, fromabout 60 nm to about 90 nm, or from about 70 nm to about 90 nm, forexample, the average diameter of the liposome particles is about 30, 40,50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.

In some embodiments, in the pharmaceutical composition formed by thedouble-stranded oligonucleotide of the present disclosure and the aboveamine-containing transfection agents, the ratio (weight/weight ratio) ofthe double-stranded oligonucleotide to total lipids, e.g., the organicamines, the helper lipids and/or the PEGylated lipids, ranges from about1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, fromabout 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 toabout 1:12, or from about 1:6 to about 1:10. For example, the ratio ofthe double-stranded oligonucleotide of the present disclosure to totallipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14,1:15, 1:16, 1:17 or 1:18 by weight.

In some embodiments, the pharmaceutical composition may be marketed witheach component being separate, and used in the form of a liquidformulation. In some embodiments, the pharmaceutical composition formedby the double-stranded oligonucleotide of the present disclosure and theabove pharmaceutically acceptable carrier may be prepared by variousknown processes, except replacing the existing double-strandedoligonucleotide with the double-stranded oligonucleotide of the presentdisclosure. In some specific embodiments, the pharmaceutical compositionmay be prepared according to the following process.

The organic amines, helper lipids and PEGylated lipids are suspended inalcohol at a molar ratio as described above and mixed homogeneously toyield a lipid solution; the alcohol is used in an amount such that theresultant lipid solution is present at a total mass concentration of 2to 25 mg/mL (e.g., 8 to 18 mg/mL). The alcohol is a pharmaceuticallyacceptable alcohol, such as an alcohol that is in liquid form at aboutroom temperature, for example, one or more of ethanol, propylene glycol,benzyl alcohol, glycerol, PEG 200, PEG 300, PEG 400, preferably ethanol.

The double-stranded oligonucleotide of the present disclosure isdissolved in a buffered salt solution to produce an aqueous solution ofthe double-stranded oligonucleotide. The buffered salt solution has aconcentration of 0.05 to 0.5 M, such as 0.1 to 0.2 M. The pH of thebuffered salt solution is adjusted to 4.0 to 5.5, such as 5.0 to 5.2.The buffered salt solution is used in an amount such that thedouble-stranded oligonucleotide is present at a concentration of lessthan 0.6 mg/ml, such as 0.2 to 0.4 mg/mL. The buffered salt may be oneor more selected from the group consisting of soluble acetate andsoluble citrate, such as sodium acetate and/or potassium acetate.

The lipid solution and the aqueous solution of the double-strandedoligonucleotide are mixed. The product obtained after mixing isincubated at a temperature of 40 to 60° C. for at least 2 minutes (e.g.,5 to 30 minutes) to produce an incubated lipid formulation. The volumeratio of the lipid solution to the aqueous solution of double-strandedoligonucleotide is 1: (2-5), such as 1:4.

The incubated lipid formulation is concentrated or diluted, purified toremove impurities, and then sterilized to obtain the pharmaceuticalcomposition of the present disclosure, which has physicochemicalparameters as follows: a pH of 6.5 to 8, an encapsulation percentage ofmore than 80%, a particle size of 40 to 200 nm, a polydispersity indexof less than 0.30, and an osmotic pressure of 250 to 400 mOsm/kg; forexample, the physicochemical parameters may be as follows: a pH of 7.2to 7.6, an encapsulation percentage of more than 90%, a particle size of60 to 100 nm, a polydispersity index of less than 0.20, and an osmoticpressure of 300 to 400 mOsm/kg.

Therein, the concentration or dilution step may be performed before,after or simultaneously with the step of impurity removal. The methodfor removing impurities may be any of various existing methods, forexample, ultrafiltration using 100 kDa hollow fiber column, PBS at pH7.4 as ultrafiltration exchange solution and the tangential flow system.The method for sterilization may be any of various existing methods,such as filtration sterilization on a 0.22 μm filter.

Oligonucleotide Conjugate

In one aspect, the present disclosure provides an oligonucleotideconjugate, which comprises the double-stranded oligonucleotide describedabove and conjugation group attached thereto.

In the context of the present disclosure, unless otherwise specified,“conjugation” refers to two or more chemical moieties each with specificfunction being linked to each other via a covalent linkage.Correspondingly, a “conjugate” refers to the compound formed bycovalently linking individual chemical moieties. Further, an“oligonucleotide conjugate” represents a compound formed by covalentlyattaching a double-stranded oligonucleotide and one or more chemicalmoieties each with specific functions. In this context, theoligonucleotide conjugate of the present disclosure is sometimesabbreviated as a “conjugate”. More specifically, in the context of thepresent disclosure, a “conjugating molecule” may be a specific compoundcapable of being conjugated to a double-stranded oligonucleotide via areaction, thereby finally forming the oligonucleotide conjugate of thepresent disclosure. The type and linking mode of the ligand iswell-known to those skilled in the art, and it typically serves thefunction of binding to the specific receptors on the surface of thetarget cell, thereby mediating delivery of the double-strandedoligonucleotide linked to the ligand into the target cell.

The conjugation group typically comprises at least one pharmaceuticallyacceptable targeting group and an optional linker. Moreover, thedouble-stranded oligonucleotide, the linker and the targeting group arelinked in succession. In one embodiment, there are 1 to 6 targetinggroups. In one embodiment, there are 2 to 4 targeting groups. Thedouble-stranded oligonucleotide molecule may be non-covalently orcovalently conjugated to the conjugation group, for example, thedouble-stranded oligonucleotide molecule is covalently conjugated to theconjugation group. The conjugation site between the double-strandedoligonucleotide and the conjugation group can be at 3′-terminal or5′-terminal of the sense strand of the double-stranded oligonucleotide,or at 5′-terminal of the antisense strand, or within the internalsequence of the double-stranded oligonucleotide. In some specificembodiments, the conjugation site between the double-strandedoligonucleotide and the conjugation group is at 3′-terminal of the sensestrand of the double-stranded oligonucleotide.

In some embodiments, the conjugation group is linked to the phosphategroup, the 2′-hydroxy group or the base of a nucleotide. In someembodiments, the conjugation group may be linked to a 3′-hydroxy groupwhen the nucleotides are linked via a 2′-5′-phosphodiester bond. Whenthe conjugation group is linked to a terminal of the double-strandedoligonucleotide, the conjugation group is typically linked to aphosphate group of a nucleotide; when the conjugation group is linked toan internal sequence of the double-stranded oligonucleotide, theconjugation group is typically linked to a ribose ring or a base. Forspecific linking modes, reference may be made to: Muthiah Manoharan et.al. siRNA conjugates carrying sequentially assembled trivalentN-acetylgalactosamine linked through nucleosides elicit robust genesilencing in vivo in hepatocytes. ACS Chemical biology, 2015,10(5):1181-7.

In some embodiments, the double-stranded oligonucleotide and theconjugation group can be linked by an acid-labile or reducible chemicalbond, and these chemical bonds can be degraded under the acidicenvironment of cell endosomes, thereby rendering the double-strandedoligonucleotide to be in free state. For non-degradable conjugationmodes, the conjugation group can be linked to the sense strand of thedouble-stranded oligonucleotide, thereby minimizing the effect ofconjugation on the activity of the double-stranded oligonucleotide.

The targeting group can be linked to the double-stranded oligonucleotidemolecule via an appropriate linker, and the appropriate linker can beselected by those skilled in the art according to the specific type ofthe targeting group. The types of these linkers and targeting groups andthe linking modes with the double-stranded oligonucleotide may be foundin the disclosures of WO2015006740A2, which is incorporated herein byreference in its entirety.

In some embodiments, when the targeting group is N-acetylgalactosamine,a suitable linker may have the following structure as shown by Formula(301):

wherein k is an integer of 1-3;

L^(A) is an amide bond-comprising chain moiety that has a structure asshown by Formula (302), each L^(A) being respectively linked to thetargeting group and the L^(C) moiety through ether bond at its twoterminals:

L^(B) is an N-acylpyrrolidine-comprising chain moiety that has astructure as shown by Formula (303), the chain moiety having a carbonylgroup at one terminal and being linked to the L^(C) moiety through anamide bond, and having an oxy-group at the other terminal and beinglinked to the double-stranded oligonucleotide through a phosphoesterbond:

L^(C) is a bivalent to tetravalent linking group based on hydroxymethylaminomethane, dihydroxymethyl aminomethane or trihydroxymethylaminomethane, L^(C) being linked to each of the L^(A) moieties throughan ether bond via oxygen atom, and being linked to L^(B) moiety throughamide bond via nitrogen atom.

In some embodiments, when n=3 and L^(C) is a tetravalent linking groupbased on trihydroxymethyl aminomethane, the oligonucleotide conjugateformed by linking N-acetylgalactosamine molecules with a double-strandedoligonucleotide molecule via -(L^(A))₃-trihydroxymethylaminomethane-L^(B)- as a linker has a structure as shown by Formula(304):

wherein the double helix structure represents a double-strandedoligonucleotide.

Likewise, the conjugation site between the double-strandedoligonucleotide and the conjugation group can be at 3′-terminal or5′-terminal of the sense strand of the double-stranded oligonucleotide,or at 5′-terminal of the antisense strand, or within the internalsequence of the double-stranded oligonucleotide.

In some specific embodiments, the 3′-terminal of the sense strand of thedouble-stranded oligonucleotide of the present disclosure is covalentlyconjugated to three N-acetylgalactosamine (GalNAc) molecules via alinker -(L^(A))₃-trihydroxymethyl aminomethane-L^(B)- to obtain anoligonucleotide conjugate in which the molar ratio of thedouble-stranded oligonucleotide molecule to the GalNAc molecule is 1:3(hereinafter referred to as (GalNAc)₃-Nu), and this oligonucleotideconjugate has a structure as shown by Formula (305):

wherein the double helix structure represents a double-strandedoligonucleotide; and the linker is linked to the 3′-terminal of thesense strand of the double-stranded oligonucleotide.

In some embodiments, when the targeting group is N-acetylgalactosamine,a suitable linker may have a structure as shown by Formula (306):

wherein 1 is an integer of 0-3;

* represents a site linked to the targeting group through ether bond onthe linker; and

# represents a site linked to the double-stranded oligonucleotide via aphosphoester bond on the linker.

In some specific embodiments, when 1=2, the oligonucleotide conjugatehas a structure as shown by Formula (307):

wherein the double helix structure represents a double-strandedoligonucleotide; and the linker is linked to the 3′-terminal of thesense strand of the double-stranded oligonucleotide.

The above conjugates can be synthesized according to the methoddescribed in detail in the prior art. For example, WO2015006740 A2described in detail the preparation of various conjugates. As anotherexample, WO2014025805A1 described the preparation method of theconjugate having the structure as shown by Formula (305). As a furtherexample, Rajeev et al., ChemBioChem 2015, 16, 903-908, described thepreparation method of the conjugate having the structure as shown byFormula (307).

In some embodiments, the conjugate has a structure as shown by Formula(308):

wherein n1 is an integer of 1-3, and n3 is an integer of 0-4;

m1, m2, and m3 independently of one another are an integer of 2-10;

R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅ independently of one another are H, orselected from the group consisting of C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl,and C₁-C₁₀ alkoxy;

R₃ is a group having a structure as shown by Formula (A59):

wherein,

E₁ is OH, SH or BH₂;

Nu is a double-stranded oligonucleotide;

R₂ is a linear alkylene of 1 to 20 carbon atoms in length, wherein oneor more carbon atoms are optionally replaced with one or more groupsselected from the group consisting of: C(O), NH, O, S, CH═N, S(O)₂,C₂-C₁₀ alkylene, C₂-C₁₀ alkynylene, C₆-C₁₀ arylene, C₃-C₁₈heterocyclylene, and C₅-C₁₀ heteroarylene, and wherein R₂ optionally hasone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₆-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₁-C₁₀ haloalkyl, —OC₁-C₁₀ alkyl,—OC₁-C₁₀ alkylphenyl, —C₁-C₁₀ alkyl-OH, —OC₁-C₁₀ haloalkyl, —SC₁-C₁₀alkyl, —SC₁-C₁₀ alkylphenyl, —C₁-C₁₀ alkyl-SH, —SC₁-C₁₀ haloalkyl, halo,—OH, —SH, —NH₂, —C₁-C₁₀ alkyl-NH₂, —N(C₁-C₁₀ alkyl)(C₁-C₁₀ alkyl),—NH(C₁-C₁₀ alkyl), cyano, nitro, —CO₂H, —C(O)OC₁-C₁₀ alkyl, —CON(C₁-C₁₀alkyl)(C₁-C₁₀ alkyl), —CONH(C₁-C₁₀ alkyl), —CONH₂, —NHC(O)(C₁-C₁₀alkyl), —NHC(O)(phenyl), —N(C₁-C₁₀ alkyl)C(O)(C₁-C₁₀ alkyl), —N(C₁-C₁₀alkyl)C(O)(phenyl), —C(O)C₁-C₁₀ alkyl, —C(O)C₁-C₁₀ alkylphenyl,—C(O)C₁-C₁₀ haloalkyl, —OC(O)C₁-C₁₀ alkyl, —SO₂(C₁-C₁₀ alkyl),—SO₂(phenyl), —SO₂(C₁-C₁₀ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₁₀ alkyl),—SO₂NH(phenyl), —NHSO₂(C₁-C₁₀ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₁₀haloalkyl);

each L₁ is independently a linear alkylene of 1 to 70 carbon atoms inlength, wherein one or more carbon atoms are optionally replaced withone or more groups selected from the group consisting of: C(O), NH, O,S, CH═N, S(O)₂, C₂-C₁₀ alkylene, C₂-C₁₀ alkynylene, C₆-C₁₀ arylene,C₃-C₁₈ heterocyclylene, and C₅-C₁₀ heteroarylene, and wherein L₁optionally has one or more substituents selected from the groupconsisting of: C₁-C₁₀ alkyl, C₆-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₁-C₁₀haloalkyl, —OC₁-C₁₀ alkyl, —OC₁-C₁₀ alkylphenyl, —C₁-C₁₀ alkyl-OH,—OC₁-C₁₀ haloalkyl, —SC₁-C₁₀ alkyl, —SC₁-C₁₀ alkylphenyl, —C₁-C₁₀alkyl-SH, —SC₁-C₁₀ haloalkyl, halo, —OH, —SH, —NH₂, —C₁-C₁₀ alkyl-NH₂,—N(C₁-C₁₀ alkyl)(C₁-C₁₀ alkyl), —NH(C₁-C₁₀ alkyl), cyano, nitro, —CO₂H,—C(O)OC₁-C₁₀ alkyl, —CON(C₁-C₁₀ alkyl)(C₁-C₁₀ alkyl), —CONH(C₁-C₁₀alkyl), —CONH₂, —NHC(O)(C₁-C₁₀ alkyl), —NHC(O)(phenyl), —N(C₁-C₁₀alkyl)C(O)(C₁-C₁₀ alkyl), —N(C₁-C₁₀ alkyl)C(O)(phenyl), —C(O)C₁-C₁₀alkyl, —C(O)C₁-C₁₀ alkylphenyl, —C(O)C₁-C₁₀ haloalkyl, —OC(O)C₁-C₁₀alkyl, —SO₂(C₁-C₁₀ alkyl), —SO₂(phenyl), —SO₂(C₁-C₁₀ haloalkyl),—SO₂NH₂, —SO₂NH(C₁-C₁₀ alkyl), —SO₂NH(phenyl), —NHSO₂(C₁-C₁₀ alkyl),—NHSO₂(phenyl), and —NHSO₂(C₁-C₁₀ haloalkyl);

M₁ represents a targeting group.

In some embodiments, L₁ may be selected from the group consisting ofgroups A1-A26 and any combination thereof, wherein the structures anddefinitions of A1-A26 are as follows:

wherein each j1 is independently an integer of 1-20;

each j2 is independently an integer of 1-20;

R′ is a C₁-C₁₀ alkyl;

Ra is selected from the group consisting of A27-A45

Rb is a C₁-C₁₀ alkyl; and

represents a site where a group is linked to the rest of the molecule.

Those skilled in the art would understand that, though L₁ is defined asa linear alkyl for convenience, but it may not be a linear group or benamed differently, such as an amine or alkenyl produced by the abovereplacement and/or substitution. For the purpose of the presentdisclosure, the length of L₁ is the number of the atoms in the chainconnecting the two attaching points. For this purpose, a ring obtainedby replacement of a carbon atom of the linear alkylene, such as aheterocyclylene or heteroarylene, is counted as one atom.

In some embodiments, the pharmaceutically acceptable targeting group maybe a conventional ligand in the field of double-stranded oligonucleotideadministration, for example, the various ligands as described inWO2009082607A2, which is incorporated herein by reference in itsentirety.

In some embodiments, each ligand is independently a ligand capable ofbinding to a cell surface receptor. In some embodiments, at least oneligand is a ligand capable of binding to a hepatocyte surface receptor.In some embodiments, at least one ligand is a ligand capable of bindingto a mammalian hepatocyte surface receptor. In some embodiments, atleast one ligand is a ligand capable of binding to a human hepatocytesurface receptor. In some embodiments, at least one ligand is a ligandcapable of binding to a hepatic surface asialoglycoprotein receptor(ASGP-R). The types of these ligands are well-known to those skilled inthe art and they typically serve the function of binding to specificreceptors on the surface of the target cell, thereby mediating deliveryof the double-stranded oligonucleotide linked to the ligand into thetarget cell.

In some embodiments, the pharmaceutically acceptable targeting group maybe any ligand that binds to the asialoglycoprotein receptor (ASGP-R) onthe surface of mammalian hepatocytes. In one embodiment, each ligand isindependently an asialoglycoprotein, such as asialoorosomucoid (ASOR) orasialofetuin (ASF).

In some embodiments, the pharmaceutically acceptable targeting group maybe selected from one or more of the ligands fromed by the followingtargeting molecules or derivatives thereof: lipophilic molecules, suchas cholesterol, bile acids, vitamins (such as vitamin E), lipidmolecules of different chain lengths; polymers, such as polyethyleneglycol; polypeptides, such as cell-penetrating peptide; aptamers;antibodies; quantum dots; saccharides, such as lactose, polylactose,mannose, galactose, N-acetylgalactosamine (GalNAc); folate; or receptorligands expressed in hepatic parenchymal cells, such asasialoglycoprotein, asialo-sugar residue, lipoproteins (such as highdensity lipoprotein, low density lipoprotein), glucagon,neurotransmitters (such as adrenaline), growth factors, transferrin andthe like.

In one embodiment, the ligand is a saccharide or its derivatives.

In some embodiments, at least one ligand is a saccharide. In someembodiments, each ligand is a saccharide. In some embodiments, at leastone ligand is a monosaccharide, polysaccharide, modified monosaccharide,modified polysaccharide, or derivatives thereof. In some embodiments, atleast one ligand may be a monosaccharide, disaccharide or trisaccharide.In some embodiments, at least one ligand is a modified saccharide. Insome embodiments, each ligand is a modified saccharide. In someembodiments, each ligand is independently selected from the groupconsisting of polysaccharides, modified polysaccharides, monosaccharidesmodified monosaccharides, polysaccharide derivatives and monosaccharidederivatives. In some embodiments, each ligand or at least one ligand maybe independently selected from the group consisting of glucose and itsderivatives, mannose and its derivatives, galactose and its derivatives,xylose and its derivatives, ribose and its derivatives, fucose and itsderivatives, lactose and its derivatives, maltose and its derivatives,arabinose and its derivatives, fructose and its derivatives, and sialicacid.

In some embodiments, each ligand may be independently selected from thegroup consisting of D-mannopyranose, L-mannopyranose, D-arabinose,D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose,L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose,β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose,α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose,α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose,α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid,galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine,N-propionylgalactosamine, N-n-butyrylgalactosamine,N-isobutyrylgalactosamine,2-amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose,2-deoxy-2-methylamino-L-glucopyranose,4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid,5-thio-β-D-glucopyranose, methyl2,3,4-tris-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-thio-β-D-galactopyranose, ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside,2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose,L-4-thioribose. Other ligand selections may be found, for example, inthe disclosure of CN105378082A, which is incorporated herein byreference in its entirety.

In some embodiments, the pharmaceutically acceptable targeting group inthe oligonucleotide conjugate may be galactose or N-acetylgalactosamine,wherein the galactose or N-acetylgalactosamine molecules can be mono-,bi-, tri-, or tetra-valent. It should be understood that the termsmono-, bi-, tri-, or tetra-valent described herein respectively meanthat the molar ratio of the double-stranded oligonucleotide molecule tothe galactose or N-acetylgalactosamine molecule in the oligonucleotideconjugate is 1:1, 1:2, 1:3 or 1:4, wherein the oligonucleotide conjugateis formed from the double-stranded oligonucleotide molecule and theconjugation group containing galactose or N-acetylgalactosamine moleculeas the targeting group. In some embodiments, the pharmaceuticallyacceptable targeting group is N-acetylgalactosamine. In someembodiments, when the double-stranded oligonucleotide of the presentdisclosure is conjugated to a conjugation group comprisingN-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalentor tetravalent. In some embodiments, when the double-strandedoligonucleotide of the present disclosure is conjugated to a conjugationgroup containing N-acetylgalactosamine, the N-acetylgalactosaminemolecule is trivalent.

M₁ represents a targeting group, of which the definitions and optionsare the same as those described above. In some embodiments, each M₁ isindependently selected from one of the ligands that have affinity to theasialoglycoprotein receptor on the surface of mammalian hepatocytes.

When M₁ is a ligand that has affinity to the asialoglycoprotein receptor(ASGP-R) on the surface of mammalian hepatocyte, in some embodiments, n1may be an integer of 1-3, and n3 may be an integer of 0-4 to ensure thatthe number of the M₁ ligand in the conjugate may be at least 2. In someembodiments, n1+n3≥2, such that the number of the M₁ ligand in theconjugate may be at least 3, thereby allowing the M₁ ligand to moreconveniently bind to the asialoglycoprotein receptor on the surface ofhepatocytes, which may facilitates the endocytosis of the conjugate intocells. Experiments have shown that when the number of the M₁ ligand isgreater than 3, the ease of binding the M₁ ligand to theasialoglycoprotein receptor on the surface of hepatocytes is notsignificantly increased. Therefore, in view of various aspects such assynthesis convenience, structure/process costs and delivery efficiency,in some embodiments, n1 is an integer of 1-2, n3 is an integer of 0-1,and n1+n3=2-3.

In some embodiments, when m1, m2, and m3 independently of one anotherare selected from an integer of 2-10, the steric mutual positions amongmany M₁ ligands may be fit for binding the M₁ ligands to theasialoglycoprotein receptor on the surface of hepatocytes. In order tomake the conjugate of the present disclosure have simpler structure,easier synthesis and/or reduced cost, in some embodiments, m1, m2 and m3independently of one another are an integer of 2-5, in some embodiments,m1=m2=m3.

Those skilled in the art would understand that when R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, and R₁₅ independently of one another are selected from H, C₁-C₁₀alkyl, C₁-C₁₀ haloalkyl, and C₁-C₁₀ alkoxy, they would not change theproperties of the conjugate of the present disclosure and could allachieve the purpose of the present disclosure. In some embodiments, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅ independently of one another are selectedfrom H, methyl and ethyl. In some embodiments, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,and R₁₅ are H.

According to the oligonucleotide conjugate of the present disclosure, R₃is a group having the structure as shown by Formula A59, wherein E₁ isOH, SH or BH₂, and considering the availability of starting materials,in some embodiments, E₁ is OH or SH.

In some embodiments, R₂ is selected to achieve the linkage between thegroup as shown by Formula (A59) and the N atom on a nitrogenousbackbone. In the context of the present disclosure, a “nitrogenousbackbone” refers to a chain structure in which the carbon atom attachedto R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, and R₁₅ and the N atoms are linked to eachother. In some embodiments, R₂ may be any linking group capable ofattaching the group as shown by Formula (A59) to the N atom on anitrogenous backbone by suitable means. In some embodiments, in the casewhere the oligonucleotide conjugate of the present disclosure isprepared by a solid phase synthesis process, R₂ group needs to have botha site linking to the N atom on the nitrogenous backbone and a sitelinking to the P atom in R₃. In some embodiments, in R₂, the sitelinking to the N atom on the nitrogenous backbone forms an amide bondwith the N atom, and the site linking to the P atom in R₃ forms aphosphoester bond with the P atom. In some embodiments, R₂ is B5, B6,B5′ or B6′:

wherein

represents the site where the group is covalently linked;

q2 is an integer of 1-10; in some embodiments, q2 is an integer of 1-5.

L₁ is used to link the M₁ ligand to the N atom on the nitrogenousbackbone, thereby providing targeting function for the oligonucleotideconjugate of the present disclosure. In some embodiments, L₁ is selectedfrom the connection combinations of one or more of Formulae A1-A26. Insome embodiments, L₁ is selected from the connection combinations of oneor more of Formulae A1, A4, A5, A6, A8, A10, A11, and A13. In someembodiments, L₁ is selected from the connection combinations of at leasttwo of Formula A1, A4, A8, A10, and A11. In some embodiments, L₁ isselected from the connection combinations of at least two of Formula A1,A8, and A10.

In some embodiments, the length of L₁ may be 3 to 25, 3 to 20, 4 to 15or 5 to 12 atoms. In some embodiments, L₁ is 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60 atoms in length.

In some embodiments, j1 is an integer of 2-10, and in some embodiments,is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, andin some embodiments, is an integer of 3-5. R′ is a C₁-C₄ alkyl, and insome embodiments, is one of methyl, ethyl, and isopropyl. Ra is one ofA27, A28, A29, A30, and A31, and in some embodiments, is A27 or A28. Rbis a C₁-C₅ alkyl, and in some embodiments, is one of methyl, ethyl,isopropyl, and butyl. In some embodiments, j1, j2, R′, Ra, and Rb ofFormulae A1-A26 are respectively selected to achieve the linkage betweenthe M₁ ligands and the N atom on the nitrogenous backbone, and to makethe steric mutual position among the M₁ ligands more suitable forbinding the M₁ ligands to the asialoglycoprotein receptor on the surfaceof hepatocytes.

In some embodiments, the oligonucleotide conjugate of the presentdisclosure has a structure as shown by Formula (403), (404), (405),(406), (407), (408), (409), (410), (411), (412), (413), (414), (415),(416), (417), (418), (419), (420), (421), or (422):

In some embodiments, the P atom in Formula A59 may be linked to anypossible position in the double-stranded oligonucleotide, for example,the P atom in Formula A59 may be linked to any nucleotide in the senseor antisense strand of the double-stranded oligonucleotide. In someembodiments, the P atom in Formula A59 is linked to any nucleotide inthe sense strand of the double-stranded oligonucleotide. In someembodiments, the P atom in Formula A59 is linked to a terminal of thesense or antisense strand of the double-stranded oligonucleotide. Insome embodiments, the P atom in Formula A59 is linked to a terminal ofthe sense strand of the double-stranded oligonucleotide. Said terminalrefers to the first 4 nucleotides counted from one terminal of the senseor antisense strand. In some embodiments, the P atom in Formula A59 islinked to either terminal of the sense or antisense strand of thedouble-stranded oligonucleotide. In some embodiments, the P atom inFormula A59 is linked to 3′ terminal of the sense strand of thedouble-stranded oligonucleotide. In the case where the P atom in FormulaA59 is linked to the above position in the sense strand of thedouble-stranded oligonucleotide, after entering into cells, theconjugate of the present disclosure can release a separate antisensestrand of the double-stranded oligonucleotide during unwinding, therebyregulating the expression of the target gene.

The P atom in Formula A59 may be linked to any possible position of anucleotide in the double-stranded oligonucleotide, for example, toposition 5′, 2′ or 3′, or to the base of the nucleotide. In someembodiments, the P atom in Formula A59 may be linked to position 2′, 3′,or 5′ of a nucleotide in the double-stranded oligonucleotide by forminga phosphodiester bond. In some embodiments, the P atom in Formula A59 islinked to an oxygen atom formed by deprotonation of 3′-hydroxy of thenucleotide at 3′ terminal of the sense strand in the double-strandedoligonucleotide, or the P atom in Formula A59 is linked to a nucleotideby substituting a hydrogen atom in 2′-hydroxy of a nucleotide of thesense strand in the double-stranded oligonucleotide, or the P atom inFormula A59 is linked to a nucleotide by substituting a hydrogen atom in5′-hydroxy of the nucleotide at 5′ terminal of the sense strand in thedouble-stranded oligonucleotide.

In the double-stranded oligonucleotide or oligonucleotide conjugate ofthe present disclosure, adjacent nucleotides are linked via aphosphodiester bond or phosphorothioate diester bond. The non-bridgingoxygen or sulfur atom in the phosphodiester bond or phosphorothioatediester bond is negatively charged, and may be present in the form ofhydroxy or sulfhydryl. Moreover, the hydrogen ion in the hydroxy orsulfhydryl may be partially or completely substituted with a cation. Thecation may be any cation, such as a metal cation, an ammonium cation NH₄⁺ or an organic ammonium cation. In order to increase solubility, in oneembodiment, the cation is selected from one or more of an alkali metalcation, an ammonium cation formed by a tertiary amine and a quaternaryammonium cation. The alkali metal ion may be K⁺ and/or Na⁺, and thecation formed by a tertiary amine may be an ammonium cation formed bytriethylamine and/or an ammonium cation formed byN,N-diisopropylethylamine. Thus, the double-stranded oligonucleotide oroligonucleotide conjugate of the present disclosure may be at leastpartially present in the form of salt. In one embodiment, non-bridgingoxygen atom or sulfur atom in the phosphodiester bond orphosphorothioate diester bond at least partly binds to sodium ion, andthus the double-stranded oligonucleotide or oligonucleotide conjugate ofthe present disclosure is present or partially present in the form ofsodium salt.

Those skilled in the art clearly know that a modified nucleotide may beintroduced into the double-stranded oligonucleotide of the presentdisclosure by a nucleoside monomer with a corresponding modification.The methods for preparing a nucleoside monomer having the correspondingmodification and the methods for introducing a modified nucleotide intoa double-stranded oligonucleotide are also well-known to those skilledin the art. All modified nucleoside monomers may be either commerciallyavailable or prepared by known methods.

Preparation of the Oligonucleotide Conjugate as Shown by Formula (308)

The oligonucleotide conjugates of the present disclosure may be preparedby any appropriate synthesis routes.

In some embodiments, the oligonucleotide conjugate as shown by Formula(308) may be prepared by the following method, comprising: successivelylinking nucleoside monomers in 3′ to 5′ direction according to thenucleotide type and sequence in the sense strand and antisense strandsof the double-stranded oligonucleotide respectively, under the conditionof phosphoramidite solid phase synthesis, wherein the step of linkingeach nucleoside monomer includes a four-step reaction of deprotection,coupling, capping, and oxidation or sulfurization; isolating the sensestrand and the antisense strand of the double-stranded oligonucleotide;and annealing, wherein the double-stranded oligonucleotide is the abovedouble-stranded oligonucleotide of the present disclosure.

Moreover, the method further comprises: contacting the compound as shownby Formula (321) with a nucleoside monomer or a nucleotide sequencelinked to a solid phase support under coupling reaction condition and inthe presence of a coupling agent, thereby linking the compound as shownby Formula (321) to the nucleotide sequence through a coupling reaction.Hereinafter, the compound as shown by Formula (321) is also called aconjugating molecule.

wherein,

R₄ is a moiety capable of binding to the double-stranded oligonucleotideof the present disclosure. In some embodiments, R₄ is a moiety capableof binding to the double-stranded oligonucleotide of the presentdisclosure via a covalent bond. In some embodiments, R₄ is a moietycomprising any functional group that may be conjugated to adouble-stranded oligonucleotide via a phosphodiester bond by a reaction;

Each S₁ is independently an M1, which is a group formed by substitutingall active hydroxyl with the group YCOO—, wherein each Y isindependently selected from the group consisting of methyl,trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl,dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl,halophenyl, and alkylphenyl.

The definitions and options of n1, n3, m1, m2, m3, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅, L₁, M₁ are respectively as described above.

R₄ is selected to achieve the linkage to the N atom on a nitrogenousbackbone and to provide a suitable reaction site for synthesizing theoligonucleotide conjugate as shown by Formula (308). In someembodiments, R₄ comprises a R₂ linking group or protected R₂ linkinggroup, and can form a functional group as shown by Formula (A59) with adouble-stranded oligonucleotide via reaction.

In some embodiments, R₄ comprises a first functional group that canreact with a group on a double-stranded oligonucleotide or a nucleosidemonomer to form a phosphite ester, and a second functional group thatcan form a covalent bond with a hydroxy group or an amino group, orcomprises a solid phase support linked via the covalent bond. In someembodiments, the first functional group is a phosphoramidite, a hydroxyor a protected hydroxy. In some embodiments, the second functional groupis a phosphoramidite, a carboxyl or a carboxylate salt. In someembodiments, the second functional group is a solid phase support linkedto the rest of the molecule via a covalent bond which is formed by ahydroxy group or an amino group. In some embodiments, the solid phasesupport is linked via a phosphoester bond, a carboxyl ester bond, or anamide bond. In some embodiments, the solid phase support is a resin.

In some embodiments, the first functional group comprises hydroxy,—OR_(k) or a group as shown by Formula (C3); the second functional groupcomprises a group as shown by Formula (C1), (C2), (C3), (C1′), or (C3′):

wherein q₁ is an integer of 1-4, X is O or NH, M⁺ is a cation, R_(k) isa hydroxy protecting group, SPS represents a solid phase support, and

represents the site where a group is covalently linked to the rest ofthe molecule.

In some embodiments, the first functional group comprises aphosphoramidite group, such as the group as shown by Formula (C3). Thephosphoramidite group can form a phosphite ester with a hydroxy at anyposition on a nucleotide (such as a 2′- or 3′-hydroxy) by couplingreaction, and the phosphite ester can form a phosphodiester bond orphosphorothioate ester bond as shown by Formula (A59) via oxidation orsulfurization, so as to conjugate the conjugating molecule to adouble-stranded oligonucleotide. Thus, even if the second functionalgroup does not exist, the compound as shown by Formula (321) will alsobe able to be conjugated to the nucleotide, without affecting theobtaining of the oligonucleotide conjugate as shown by Formula (308).Under such circumstances, after obtaining a sense or antisense strand ofthe double-stranded oligonucleotide by a method such as phosphoramiditesolid phase synthesis, the compound as shown by Formula (321) is reactedwith a hydroxy on the terminal nucleotide of the nucleotide sequence,and the resultant phosphite ester forms a phosphodiester bond orphosphorothioate bond by a subsequent oxidation or sulfurization,thereby conjugating the compound as shown by Formula (321) to adouble-stranded oligonucleotide.

In some embodiments, the first functional group comprises a protectedhydroxy group. In some embodiments, the second functional groupcomprises a group that can react with a solid phase support to provide aconjugating molecule comprising the solid phase support. In someembodiments, the second functional group comprises a carboxyl, acarboxylate or a phosphoramidite, such as the functional group as shownby Formula (C1), (C2) or (C3). When the second functional groupcomprises a carboxyl or a carboxylate, the compound as shown by Formula(321) can react via an esterification or an amidation reaction with ahydroxy or an amino group on a solid phase support such as a resin, toform a conjugating molecule comprising a solid phase support linked viaa carboxylate ester bond or an amide bond. When the second functionalgroup comprises a phosphoramidite functional group, the compound asshown by Formula (321) can be coupled with a hydroxy group on auniversal solid phase support, such as a resin, and by oxidation, form aconjugating molecule comprising a solid phase support linked via aphosphodiester bond. Subsequently, starting from the above productlinked to a solid phase support, the nucleoside monomers are linkedsequentially by a phosphoramidite solid phase synthesis method, therebyobtaining a sense or antisense strand of the double-strandedoligonucleotide linked to the conjugation group. During the solid phasephosphoramidite synthesis, the first functional group is deprotected,and then coupled with a phosphoramidite group on a nucleoside monomerunder coupling reaction condition.

In some embodiments, the first functional group comprises a hydroxy or aprotected hydroxy group, and the second functional group comprises asolid phase support linked via a carboxylate ester bond, a amide bond ora phosphoester bond as shown by Formula (C1′) or (C3′). Under suchcircumstances, starting from the compound as shown by Formula (321) inplace of the solid phase support, the nucleoside monomers are linkedsequentially by a phosphoramidite solid phase synthesis method, therebyobtaining a sense or antisense strand of the double-strandedoligonucleotide linked to a conjugation group.

In some embodiments, the carboxylate may be expressed as —COO⁻M⁺,wherein M⁺ is a cation such as one of a metal cation, an ammonium cationNH₄ ⁺ and an organic ammonium cation. In one embodiment, the metalcation may be an alkali metal cation, such as K⁺ or Na⁺. In order toincrease solubility and facilitate the reaction, in one embodiment, theorganic ammonium cation is an ammonium cation formed by a tertiaryamine, or a quaternary ammonium cation, such as an ammonium cationformed by triethylamine or N,N-diisopropylethylamine. In someembodiments, the carboxylate is a triethylamine carboxylate or anN,N-diisopropylethylamine carboxylate.

In some embodiments, R₄ comprises a structure as shown by Formula (B9),(B10), (B9′), (B10′), (B11), (B12), (B11′) or (B12′):

wherein q₁ is an integer of 1-4, q2 is an integer of 1-10, X is O or NH,M⁺ is a cation, R_(k) is a hydroxy protecting group, SPS represents asolid phase support, and

represents the site where a group is covalently linked to the rest ofthe molecule. In some embodiments, q₁ is 1 or 2. In some embodiments, q₂is an integer of 1-5. In some embodiments, R₄ comprises a structure asshown by Formula (B9) or (B10). In some embodiments, R₄ comprises astructure as shown by Formula (B11) or (B12).

In some embodiments, R_(k) is one or more of Tr (trityl), MMTr(4-methoxytrityl), DMTr (4,4′-dimethoxytrityl), and TMTr(4,4′,4″-trimethoxytrityl). In some embodiments, R_(k) may be DMTr,i.e., 4,4′-dimethoxytrityl.

L₁ is a linear alkylene of 1 to 70 carbon atoms in length, wherein oneor more carbon atoms are optionally replaced with one or more groupsselected from the group consisting of: C(O), NH, O, S, CH═N, S(O)₂,C₂-C₁₀ alkylene, C₂-C₁₀ alkynylene, C₆-C₁₀ arylene, C₃-C₁₈heterocyclylene, and C₅-C₁₀ heteroarylene, and wherein L₁ optionally hasone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₆-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₁-C₁₀ haloalkyl, —OC₁-C₁₀ alkyl,—OC₁-C₁₀ alkylphenyl, —C₁-C₁₀ alkyl-OH, —OC₁-C₁₀ haloalkyl, —SC₁-C₁₀alkyl, —SC₁-C₁₀ alkylphenyl, —C₁-C₁₀ alkyl-SH, —SC₁-C₁₀ haloalkyl, halo,—OH, —SH, —NH₂, —C₁-C₁₀ alkyl-NH₂, —N(C₁-C₁₀ alkyl)(C₁-C₁₀ alkyl),—NH(C₁-C₁₀ alkyl), cyano, nitro, —CO₂H, —C(O)OC₁-C₁₀ alkyl, —CON(C₁-C₁₀alkyl)(C₁-C₁₀ alkyl), —CONH(C₁-C₁₀ alkyl), —CONH₂, —NHC(O)(C₁-C₁₀alkyl), —NHC(O)(phenyl), —N(C₁-C₁₀ alkyl)C(O)(C₁-C₁₀ alkyl), —N(C₁-C₁₀alkyl)C(O)(phenyl), —C(O)C₁-C₁₀ alkyl, —C(O)C₁-C₁₀ alkylphenyl,—C(O)C₁—C₁₀ haloalkyl, —OC(O)C₁-C₁₀ alkyl, —SO₂(C₁-C₁₀ alkyl),—SO₂(phenyl), —SO₂(C₁-C₁₀ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₁₀ alkyl),—SO₂NH(phenyl), —NHSO₂(C₁-C₁₀ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₁₀haloalkyl).

In some embodiments, L₁ is used to link the M1 ligand to the N atom onthe nitrogenous backbone, thereby providing liver targeting function forthe oligonucleotide conjugate. In some embodiments, L₁ comprises any oneof Formulae A1-A26, or the combination thereof.

According to the embodiments described above, those skilled in the artwould easily understand that as compared with the well-knownphosphoramidite solid phase synthesis methods in the art, anoligonucleotide conjugate in which a conjugating molecule is linked toany possible position of the nucleotide sequence can be obtained throughthe above first functional group and an optional second functionalgroup. For example, the conjugating molecule is linked to a terminal ofthe nucleotide sequence or to either terminal of the nucleotidesequence. Correspondingly, unless otherwise specified, in the followingdescription regarding conjugate preparation, when referring to thereactions such as “deprotection”, “coupling”, “capping”, “oxidation”,“sulfurization”, it will be understood that the reaction conditions andagents involved in the well-known phosphoramidite solid phase synthesismethods in the art would also apply to these reactions. Exemplaryreaction conditions and agents will be described in detail hereinafter.

In some embodiments, each S₁ is independently an M₁. In someembodiments, each S₁ is independently a group formed by protecting atleast one active hydroxyl in M1 with a hydroxyl protecting group. Insome embodiments, S₁ is independently a group formed by protecting allactive hydroxyls in M1 with hydroxyl protecting groups. In someembodiments, any hydroxyl protecting group known to those skilled in theart may be used to protect the active hydroxyl on M₁. In someembodiments, the protected hydroxy is expressed as the formula YCOO—,wherein each Y is independently selected from the group consisting ofC₁-C₁₀ alkyl and C₆-C₁₀ aryl, which is optionally substituted with oneor more substituents selected from the group consisting of halo andC₁-C₆ alkyl. In some embodiments, each Y is independently selected fromthe group consisting of methyl, trifluoromethyl, difluoromethyl,monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl,ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C₁-C₆ alkylphenyl.

In some embodiments, each S₁ is independently selected from the groupconsisting of Formulae A46-A54:

In some embodiments, S₁ is Formula A49 or A50.

In some embodiments, each Y is independently selected from one ofmethyl, trifluoromethyl, difluoromethyl, monofluoromethyl,trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl,isopropyl, phenyl, halophenyl, and alkylphenyl. In some embodiments, Yis methyl.

As mentioned previously, the method for preparing the oligonucleotideconjugate of the present disclosure further comprises the followingstep: synthesizing the other strand of the double-strandedoligonucleotide (for example, when a sense strand of the double-strandedoligonucleotide linked to a conjugation group is synthesized in theabove steps, the method further comprises synthesizing an antisensestrand of the double-stranded oligonucleotide by the solid phasesynthesis method, and vice versa), isolating the sense strand and theantisense strand, and annealing. In particular, in the isolation step,the solid phase support linked to the nucleotide sequence and/orconjugation group is cleaved, the necessary protecting group is removed(in this case, each S₁ group in the compound as shown by Formula (321)is converted to the corresponding M₁ ligand), a sense strand (orantisense strand) of the double-stranded oligonucleotide linked to theconjugation group and the corresponding antisense strand (or sensestrand) are obtained, and the sense strand and the antisense strand areannealed to form a double-stranded RNA structure, thereby obtaining anoligonucleotide conjugate as shown by Formula (308).

In some embodiments, the method for preparing the oligonucleotideconjugate comprises the following steps: contacting the compound asshown by Formula (321) with the first nucleoside monomer at 3′ terminalof the sense or antisense strand under coupling reaction condition inthe presence of a coupling agent, thereby linking the compound as shownby Formula (321) to the first nucleotide in the sequence; successivelylinking nucleoside monomers in 3′ to 5′ direction to synthesize thesense or antisense strand of the double-stranded oligonucleotideaccording to the desired nucleotide type and sequence of the sense orantisense strand, under the condition of phosphoramidite solid phasesynthesis; wherein the compound of Formula (321) is a compound in whichR₄ comprises a first functional group comprising a protected hydroxy anda second functional group comprising a group as shown by Formula (C1′)or (C3′), and the compound of Formula (321) is deprotected before linkedto the first nucleoside monomer; and the linking of each nucleosidemonomer comprises a four-step reaction of deprotection, coupling,capping, and oxidation or sulfurization; thus obtaining a sense orantisense strand of nucleic acid linked to the conjugation group;successively linking nucleoside monomers in 3′ to 5′ direction tosynthesize the sense or antisense strand of nucleic acid according tothe nucleotide type and sequence of the sense or antisense strand, underthe condition of phosphoramidite solid phase synthesis; wherein thelinking of each nucleoside monomer includes a four-step reaction ofdeprotection, coupling, capping, and oxidation or sulfurization;removing the protecting groups and cleaving the solid phase support;isolating and purifying the sense strand and the antisense strand ofnucleic acid; and annealing.

In some embodiments, the method for preparing the oligonucleotideconjugate comprises the following steps: successively linking nucleosidemonomers in 3′ to 5′ direction to synthesize the sense strand or theantisense strand according to the nucleotide type and sequence of thesense or antisense strand in the double-stranded oligonucleotide;wherein the linking of each nucleoside monomer comprises a four-stepreaction of deprotection, coupling, capping, and oxidation orsulfurization, thus obtaining a sense strand linked to the solid phasesupport and an antisense strand linked to the solid phase support;contacting the compound as shown by Formula (321) with the sense strandlinked to the solid phase support or the antisense strand linked to thesolid phase support under coupling reaction condition in the presence ofa coupling agent, thereby linking the compound as shown by Formula (321)to the sense strand or the antisense strand; wherein the compound ofFormula (321) is a compound in which R₄ comprises a phosphoramiditegroup as the first functional group; removing the protecting groups andcleaving the solid phase support; respectively isolating and purifyingthe sense or antisense strand of the double-stranded oligonucleotide;and annealing; wherein the sense or antisense strand of thedouble-stranded oligonucleotide is linked to a conjugation group.

In some embodiments, the P atom in formula A59 is linked to the 3′terminal of the sense strand of the double-stranded oligonucleotide, andthe method for preparing the oligonucleotide conjugate of the presentdisclosure comprises:

(1) removing the hydroxyl protecting group R_(k) in the compound ofFormula (321) (wherein the compound of Formula (321) is a compound inwhich R₄ comprises a first functional group and a second function group,wherein the first functional group comprises a protected hydroxy OR_(k),and the second function group has a structure as shown by Formula (C1′)or (C3′)); contacting the deprotected product with a nucleoside monomerto obtain a nucleoside monomer linked to a solid phase support via theconjugation group, under coupling reaction condition in the presence ofa coupling agent;

(2) starting from the nucleoside monomer linked to a solid phase supportvia the conjugating molecule, synthesizing a sense strand of thedouble-stranded oligonucleotide in 3′ to 5′ direction by aphosphoramidite solid phase synthesis method;

(3) synthesizing an antisense strand of the double-strandedoligonucleotide by a phosphoramidite solid phase synthesis method; and

(4) isolating the sense strand and the antisense strand of thedouble-stranded oligonucleotide and annealing the same to obtain theoligonucleotide conjugate of the present disclosure;

wherein in step (1), the method for removing the protecting group R_(k)in the compound of Formula (321) comprises contacting the compound ofFormula (321) with a deprotection agent under deprotection condition.The deprotection condition comprises a temperature of 0-50° C., and insome embodiments, 15-35° C., and a reaction time of 30-300 seconds, andin some embodiments, 50-150 seconds. The deprotection agent may beselected from one or more of trifluoroacetic acid, trichloroacetic acid,dichloroacetic acid, and monochloroacetic acid, and in some embodiments,the deprotection agent is dichloroacetic acid. The molar ratio of thedeprotection agent to the compound as shown by Formula (321) may be 10:1to 1000:1, and in some embodiments, 50:1 to 500:1.

The coupling reaction condition and the coupling agent may be anyconditions and agents suitable for the above coupling reaction. In someembodiments, the same condition and agent as those of the couplingreaction in the solid phase synthesis method can be used.

In some embodiments, the coupling reaction condition comprises areaction temperature of 0-50° C., and in some embodiments, 15-35° C. Themolar ratio of the compound of Formula (321) to the nucleoside monomermay be 1:1 to 1:50, and in some embodiments, 1:2 to 1:5. The molar ratioof the compound of Formula (321) to the coupling agent may be 1:1 to1:50, and in some embodiments, 1:3 to 1:10. The reaction time may be200-3000 seconds, and in some embodiments, 500-1500 seconds. Thecoupling agent may be selected from one or more of 1H-tetrazole,5-ethylthio-1H-tetrazole and 5-benzylthio-1H-tetrazole, and in someembodiments, is 5-ethylthio-1H-tetrazole. The coupling reaction may beperformed in an organic solvent. The organic solvent may be selectedfrom one or more of anhydrous acetonitrile, anhydrous DMF and anhydrousdichloromethane, and in some embodiments, is anhydrous acetonitrile. Theamount of the organic solvent may be 3-50 L/mol, and in someembodiments, 5-20 L/mol, with respect to the compound as shown byFormula (321).

In step (2), a sense strand S of the oligonucleotide conjugate issynthesized in 3′ to 5′ direction by the phosphoramidite solid phasesynthesis method, starting from the nucleoside monomer linked to a solidphase support via a conjugating molecule prepared in the above steps. Inthis case, the conjugation group is linked to the 3′ terminal of theresultant sense strand.

Other conditions for the solid phase synthesis in steps (2) and (3),including the deprotection condition for the nucleoside monomer, thetype and amount of the deprotection agent, the coupling reactioncondition, the type and amount of the coupling agent, the cappingreaction condition, the type and amount of the capping agent, theoxidation reaction condition, the type and amount of the oxidationagent, the sulfurization reaction condition, and the type and amount ofthe sulfurization agent, adopt various conventional agents, amounts, andconditions in the art.

In some embodiments, for example, the solid phase synthesis in steps (2)and (3) can use the following conditions:

The deprotection condition for the nucleoside monomer comprises areaction temperature of 0-50° C., and in some embodiments, 15-35° C.,and a reaction time of 30-300 seconds, and in some embodiments, 50-150seconds. The deprotection agent may be selected from one or more oftrifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, andmonochloroacetic acid, and in some embodiments, is dichloroacetic acid.The molar ratio of the deprotection agent to the protecting group4,4′-dimethoxytrityl on the solid phase support may be 2:1 to 100:1, andin some embodiments, is 3:1 to 50:1.

The coupling reaction condition comprises a reaction temperature of0-50° C., and in some embodiments, 15-35° C. The molar ratio of thenucleic acid sequence linked to the solid phase support to thenucleoside monomer may be 1:1 to 1:50, and in some embodiments, is 1:5to 1:15. The molar ratio of the nucleic acid sequence linked to thesolid phase support to the coupling agent may be 1:1 to 1:100, and insome embodiments, is 1:50 to 1:80. The selection of the reaction timeand the coupling agent can be same as above.

The capping reaction condition comprises a reaction temperature of 0-50°C., and in some embodiments, 15-35° C., and a reaction time of 5-500seconds, and in some embodiments, 10-100 seconds. The selection of thecapping agent can be same as above. The molar ratio of the total amountof the capping agent to the nucleic acid sequence linked to the solidphase support may be 1:100 to 100:1, and in some embodiments, is 1:10 to10:1. In the case where the capping agent uses equimolar aceticanhydride and N-methylimidazole, the molar ratio of acetic anhydride,N-methylimidazole, and the nucleic acid sequence linked to the solidphase support may be 1:1:10-10:10:1, and in some embodiments, is1:1:2-2:2:1.

The oxidation reaction condition comprises a reaction temperature of0-50° C., and in some embodiments, 15-35° C., and a reaction time of1-100 seconds, and in some embodiments, 5-50 seconds. In someembodiments, the oxidation agent is iodine (in some embodiments providedas iodine water). The molar ratio of the oxidation agent to the nucleicacid sequence linked to the solid phase support in the coupling step maybe 1:1 to 100:1, and in some embodiments, is 5:1 to 50:1. In someembodiments, the oxidation reaction is performed in a mixed solvent inwhich the ratio of tetrahydrofuran:water:pyridine is 3:1:1-1:1:3. Thesulfurization reaction condition comprises a reaction temperature of0-50° C., and in some embodiments, 15-35° C., and a reaction time of50-2000 seconds, and in some embodiments, 100-1000 seconds. In someembodiments, the sulfurization agent is xanthane hydride. The molarratio of the sulfurization agent to the nucleic acid sequence linked tothe solid phase support in the coupling step may be 10:1 to 1000:1, andin some embodiments, is 10:1 to 500:1. In some embodiments, thesulfurization reaction is performed in a mixed solvent in which theratio of acetonitrile:pyridine is 1:3-3:1.

The method further comprises isolating the sense strand and theantisense strand of the double-stranded oligonucleotide after linkingall nucleoside monomers and before the annealing. Methods for isolationare well-known to those skilled in the art and generally comprisecleaving the synthesized nucleotide sequence from the solid phasesupport, removing protecting groups on the bases, phosphate groups andligands, purifying and desalting.

The conventional cleavage and deprotection methods in the synthesis ofdouble-stranded oligonucleotides can be used to cleave the synthesizednucleotide sequence from the solid phase support, and remove theprotecting groups on the bases, phosphate groups and ligands. Forexample, contacting the resultant nucleotide sequence linked to thesolid phase support with concentrated aqueous ammonia; duringdeprotection, the protecting group YCOO⁻ in groups A46-A54 is convertedto a hydroxyl group, and thus the S₁ groups are converted tocorresponding M₁ groups, providing the conjugate as shown by Formula(308); wherein the concentrated aqueous ammonia may be aqueous ammoniaof a concentration of 25-30% by weight. The amount of the concentratedaqueous ammonia may be 0.2 ml/μmol-0.8 ml/μmol with respect to thetarget double-stranded oligonucleotide.

When there is at least one 2′-TBDMS protection on the synthesizednucleotide sequence, the method further comprises contacting thenucleotide sequence removed from the solid phase support withtriethylamine trihydrofluoride to remove the 2′-TBDMS protection. Here,the resultant target double-stranded oligonucleotide comprises thecorresponding nucleoside having free 2′-hydroxy. The amount of puretriethylamine trihydrofluoride may be 0.4 ml/μmol-1.0 ml/μmol withrespect to the target double-stranded oligonucleotide. As such, theoligonucleotide conjugate as shown by Formula (308) may be obtained.

Methods for purification and desalination are well-known to thoseskilled in the art. For example, nucleic acid purification may beperformed using a preparative ion chromatography purification columnwith a gradient elution of NaBr or NaCl; after collection andcombination of the product, the desalination may be performed using areverse phase chromatography purification column.

The non-bridging oxygen or sulfur atom in the phosphodiester bond orphosphorothioate diester bond between the nucleotides in the resultantoligonucleotide conjugate substantially binds to a sodium ion, and theoligonucleotide conjugate is substantially present in the form of asodium salt. The well-known ion-exchange methods may be used, in whichthe sodium ion may be replaced with hydrogen ion and/or other cations,thereby providing other forms of oligonucleotide conjugates. The cationsare as described above.

During synthesis, the purity and molecular weight of the nucleic acidsequence may be determined at any time, in order to better control thesynthesis quality. Such determination methods are well-known to thoseskilled in the art. For example, the purity of the nucleic acid may bedetermined by ion exchange chromatography, and the molecular weight maybe determined by liquid chromatography-mass spectrometry (LC-MS).

Methods for annealing are also well-known to those skilled in the art.For example, the synthesized sense strand (S strand) and antisensestrand (AS strand) may be simply mixed in water for injection at anequimolar ratio, heated to 70-95° C., and then cooled at roomtemperature to form a double-stranded structure via hydrogen bond.Hence, the oligonucleotide conjugates of the present disclosure can beobtained.

After obtaining the conjugate of the present disclosure, in someembodiments, the oligonucleotide conjugate thus synthesized can also becharacterized by the means such as molecular weight detection using themethods such as LC-MS, to confirm that the synthesized oligonucleotideconjugate is the designed oligonucleotide conjugate of interest, and thesequence of the synthesized double-stranded oligonucleotide is thesequence of the desired double-stranded oligonucleotide, for example, isone of the sequences as listed in Table 1.

The compound as shown by Formula (321) may be prepared by the followingmethod comprising: contacting a compound as shown by Formula (313) witha cyclic anhydride in an organic solvent under esterification reactioncondition in the presence of a base and an esterification catalyst;isolating the compound as shown by Formula (321) by ion exchange:

wherein the definitions and options of n1, n3, m1, m2, m3, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄, R₁₅, L₁, S₁ are respectively as described above;

R₆ is a group for providing R₄ of Formula (321). In some embodiments,for example, R₆ has a structure as shown by Formula (A61):

wherein,

R_(i) is any group capable of linking to the N atom on the nitrogenousbackbone, linking to R_(k)O and linking to a free hydroxy group; R_(k)is a hydroxy protecting group. In this case, a compound as shown byFormula (321) is obtained, wherein R₄ comprises a first functional groupas a hydroxy protecting group and a second functional group comprising agroup as shown by Formula (C1) or (C2).

The esterification reaction condition includes a reaction temperature of0-100° C. and a reaction time of 8-48 hours. In some embodiments, theesterification reaction condition comprises a reaction temperature of10-40° C. and a reaction time of 20-30 hours.

In some embodiments, the organic solvent comprises one or more of anepoxy solvent, an ether solvent, an haloalkane solvent, dimethylsulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In someembodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. Insome embodiments, the ether solvent is diethyl ether and/or methyltertbutyl ether. In some embodiments, the haloalkane solvent is one ormore of dichloromethane, trichloromethane and 1,2-dichloroethane. Insome embodiments, the organic solvent is dichloromethane. The amount ofthe organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol,with respect to the compound as shown by Formula (313).

In some embodiments, the cyclic anhydride is one of succinic anhydride,glutaric anhydride, adipic anhydride or pimelic anhydride, and in someembodiments, the cyclic anhydride is succinic anhydride. The molar ratioof the cyclic anhydride to the compound as shown by Formula (313) is 1:1to 10:1, and in some embodiments, 2:1 to 5:1.

The esterification catalyst may be any catalyst capable of catalyzingesterification, such as 4-dimethylaminopyridine. The molar ratio of thecatalyst to the compound as shown by Formula (313) is 1:1 to 10:1, andin some embodiments, is 2:1 to 5:1.

In some embodiments, the base may be any inorganic base, organic base orcombination thereof. Considering solubility and product stability, thebase is an organic base of tertiary amine. In some embodiments, theorganic base of tertiary amine is triethylamine orN,N-diisopropylethylamine. The molar ratio of the organic base oftertiary amine to the compound as shown by Formula (313) is 1:1 to 20:1,and in some embodiments, is 3:1 to 10:1.

The ion exchange serves the function of converting the compound as shownby Formula (321) into a desired form of carboxylic acid or carboxylicsalt and the methods of ion exchange are well-known to those skilled inthe art. The above conjugating molecule in which the cation is M⁺ may beobtained by using suitable ion exchange solution and ion exchangecondition, which is not described here in detail. In some embodiments, atriethylamine phosphate solution is used in the ion exchange reaction.In some embodiments, the concentration of the triethylamine phosphatesolution is 0.2-0.8 M. In some embodiments, the concentration of thetriethylamine phosphate solution is 0.4-0.6 M. In some embodiments, theamount of the triethylamine phosphate solution is 3-6 L/mol, and infurther embodiment, 4-5 L/mol, with respect to the compound as shown byFormula (313).

The compound as shown by Formula (321) may be isolated from the reactionmixture using any suitable isolation methods. In some embodiments, thecompound as shown by Formula (321) may be isolated by removal of solventvia evaporation followed by chromatography, for example, using thefollowing chromatographic conditions for the isolation: (1) normal phasepurification: 200-300 mesh silica gel filler, gradient elution of 1 wt ‰triethylamine in dichloromethane:methanol=100:18-100:20; or (2) reversephase purification: C18 and C8 reverse phase filler, gradient elution ofmethanol:acetonitrile=0.1:1-1:0.1. In some embodiments, the solvent maybe directly removed to obtain a crude product of the compound as shownby Formula (321), which may be directly used in subsequent reactions.

In some embodiments, the method for preparing the compound as shown byFormula (321) further comprises: contacting the product obtained fromthe above ion exchanging reaction with a solid phase support with aminoor hydroxy groups in an organic solvent under condensation reactioncondition in the presence of a condensing agent and an organic base oftertiary amine.

In this case, a compound as shown by Formula (321) is obtained, whereinR₄ comprises a first functional group comprising a hydroxy protectinggroup and a second functional group having a structure as shown byFormula (C1′).

The solid phase support is one of the supports used in solid phasesynthesis of double-stranded oligonucleotides, some of which arewell-known to those skilled in the art. For example, the solid phasesupport may be selected from the solid phase supports containing anactive hydroxy or amino functional group. In some embodiments, the solidphase support is an amino or hydroxy resin. In some embodiments, theamino or hydroxy resin has the following parameters: particle size of100-400 mesh, and surface amino or hydroxy loading of 0.2-0.5 mmol/g.The ratio of the compound as shown by Formula (321) to the solid phasesupport is 10 μmol compound per gram of solid phase support (μmol/g) to400 μmol/g. In some embodiments, the ratio of compound of Formula (321)to the solid phase support is 50 μmol/g to 200 μmol/g.

The organic solvent may be any suitable solvent or mixed solvents knownto those skilled in the art. In some embodiments, the organic solvent isone or more of acetonitrile, an epoxy solvent, an ether solvent, anhaloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, andN,N-diisopropylethylamine. In some embodiments, the epoxy solvent isdioxane and/or tetrahydrofuran; the ether solvent is diethyl etherand/or methyl tertbutyl ether; the haloalkane solvent is one or more ofdichloromethane, trichloromethane and 1,2-dichloroethane. In someembodiments, the organic solvent is acetonitrile. The amount of theorganic solvent is 20-200 L/mol, in some embodiments, 50-100 L/mol, withrespect to the compound as shown by Formula (321).

In some embodiments, the condensing agent may bebenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate,3-diethoxyphosphoryl-1,2,3-benzotrizin-4(3H)-one and/orO-benzotriazol-tetramethyluronium hexafluorophosphate. In someembodiments, the condensing agent is O-benzotriazol-tetramethyluroniumhexafluorophosphate. The molar ratio of the condensing agent to thecompound as shown by Formula (321) is 1:1 to 20:1, and in furtherembodiments, 1:1 to 5:1.

In some embodiments, the organic base of tertiary amine is triethylamineand/or N,N-diisopropylethylamine, and in some embodiments,N,N-diisopropylethylamine. The molar ratio of the organic base oftertiary amine to the compound as shown by Formula (321) is 1:1 to 20:1,and in some embodiments, 1:1 to 5:1.

In some embodiments, the method for preparing the compound as shown byFormula (321) further comprises: contacting the resultant condensationproduct with a capping agent and an acylation catalyst in an organicsolvent under capping reaction condition, and isolating the compound asshown by Formula (321). The capping reaction is used to remove anyactive functional group that does not completely react, so as to avoidproducing unnecessary by-products in subsequent reactions. The cappingreaction condition comprises a reaction temperature of 0-50° C., and insome embodiments, 15-35° C., and a reaction time of 1-10 hours, and insome embodiments, 3-6 hours. The capping agent may be a capping agentused in solid phase synthesis of a nucleic acid, which is well known tothose skilled in the art.

In some embodiments, the capping agent is composed of capping agent A(capA) and capping agent B (capB). The capA is N-methylimidazole, and insome embodiments, provided as a mixed solution of N-methylimidazole inpyridine/acetonitrile, wherein the volume ratio of pyridine toacetonitrile is 1:10 to 1:1, and in some embodiments, 1:3 to 1:1. Insome embodiments, the ratio of the total volume of pyridine andacetonitrile to the volume of N-methylimidazole is 1:1 to 10:1, and insome embodiments, 3:1 to 7:1. In some embodiments, the capB is aceticanhydride. In some embodiments, the capB is provided as a solution ofacetic anhydride in acetonitrile, wherein the volume ratio of aceticanhydride to acetonitrile is 1:1 to 1:10, and in further embodiments,1:2 to 1:6.

In some embodiments, the ratio of the volume of the mixed solution ofN-methylimidazole in pyridine/acetonitrile to the mass of the compoundof Formula (321) is 5 ml/g-50 ml/g, and in some embodiments, 15 ml/g-30ml/g. The ratio of the volume of the solution of acetic anhydride inacetonitrile to the mass of the compound of Formula (321) is 0.5 ml/g-10ml/g, and in some embodiments, 1 ml/g-5 ml/g.

In some embodiments, the capping agent comprises equimolar aceticanhydride and N-methylimidazole. In some embodiments, the organicsolvent is one or more of acetonitrile, an epoxy solvent, an ethersolvent, an haloalkane solvent, dimethyl sulfoxide,N,N-dimethylformamide, and N,N-diisopropylethylamine. In someembodiments, the organic solvent is acetonitrile. In some embodiments,the amount of the organic solvent is 10-50 L/mol, and in someembodiments, 5-30 L/mol, with respect to the compound as shown byFormula (321).

In some embodiments, the acylation catalyst may be selected from anycatalyst that may be used for esterification condensation or amidationcondensation, such as alkaline heterocyclic compounds. In someembodiments, the acylation catalyst is 4-dimethylaminopyridine. The massratio of the catalyst to the compound as shown by Formula (321) may be0.001:1 to 1:1, and in some embodiments, 0.01:1 to 0.1:1.

In some embodiments, the compound as shown by Formula (321) may beisolated from the reaction mixture by any suitable methods. In someembodiments, the compound of Formula (321) may be obtained by thoroughlywashing with an organic solvent and filtering to remove unreactedreactants, excess capping agent and other impurities, wherein theorganic solvent is selected from acetonitrile, dichloromethane, ormethanol. In some embodiments, the organic solvent is acetonitrile.

In some embodiments, the preparation of the conjugating molecule asshown by Formula (321) comprises contacting a compound as shown byFormula (313) with a phosphorodiamidite in an organic solvent undercoupling reaction condition in the presence of a coupling agent, andisolating the compound as shown by Formula (321). In this case, acompound as shown by Formula (321) is obtained, where R₄ comprises afirst functional group comprising a hydroxy protecting group and asecond functional group having a structure as shown by Formula (C3).

In some embodiments, the coupling reaction condition comprises areaction temperature of 0-50° C., such as 15-35° C. The molar ratio ofthe compound of Formula (313) to the phosphorodiamidite may be 1:1 to1:50, such as 1:5 to 1:15. The molar ratio of the compound of Formula(313) to the coupling agent may be 1:1 to 1:100, such as 1:50 to 1:80.The reaction time may be 200-3000 seconds, such as 500-1500 seconds. Thephosphorodiamidite may be, for example,bis(diisopropylamino)(2-cyanoethoxy)phosphine, which may be commerciallyavailable or synthesized according to well-known methods in the art. Thecoupling agent is selected from one or more of 1H-tetrazole,5-ethylthio-1H-tetrazole and 5-benzylthio-1H-tetrazole, such as5-ethylthio-1H-tetrazole. The coupling reaction may be performed in anorganic solvent. In some embodiments, the organic solvent is selectedfrom one or more of anhydrous acetonitrile, anhydrous DMF and anhydrousdichloromethane, such as anhydrous acetonitrile. The amount of theorganic solvent may be 3-50 L/mol, such as 5-20 L/mol, with respect tothe compound as shown by Formula (313). By performing the couplingreaction, the hydroxy group in the compound (313) reacts with thephosphorodiamidite to form a phosphoramidite group. In some embodiments,the solvent may be directly removed to obtain a crude product of thecompound as shown by Formula (321), which may be directly used insubsequent reactions.

In some embodiments, the method for preparing the compound as shown byFormula (321) further comprises: contacting the isolated product with asolid phase support with hydroxy groups in an organic solvent undercoupling reaction condition in the presence of a coupling agent,followed by capping, oxidation, and isolation, to obtain the compound asshown by Formula (321), where R₄ a first functional group comprising ahydroxy protecting group and a second functional group having astructure as shown by Formula (C3′).

In some embodiments, the solid phase support is a well-known support inthe art for solid phase synthesis of a nucleic acid, such as adeprotected commercially available universal solid phase support, suchas NittoPhase® HL UnyLinker™ 300 Oligonucleotide Synthesis Support,Kinovate Life Sciences, as shown by Formula B80:

A deprotection reaction is well-known in the art. In some embodiments,the deprotection condition comprises a temperature of 0-50° C., such as15-35° C., and a reaction time of 30-300 seconds, such as 50-150seconds. The deprotection agent may be selected from one or more oftrifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, andmonochloroacetic acid. In some embodiments, the deprotection agent isdichloroacetic acid. The molar ratio of the deprotection agent to theprotecting group -DMTr (4,4′-dimethoxytrityl) on the solid phase supportmay be 2:1 to 100:1, such as 3:1 to 50:1. By such deprotection, reactivefree hydroxy groups are obtained on the surface of the solid phasesupport, for facilitating the subsequent coupling reaction.

The coupling reaction condition and the coupling agent may be selectedas above. By such a coupling reaction, the free hydroxy groups formed inthe deprotection reaction reacts with the phosphoramidite groups, so asto form a phosphite ester linkage.

In some embodiments, the capping reaction condition comprises atemperature of 0-50° C., such as 15-35° C., and a reaction time of 5-500seconds, such as 10-100 seconds. The capping reaction is performed inthe presence of a capping agent. The selection and amount of the cappingagent are as above.

The oxidation reaction condition may comprise a temperature of 0-50° C.,such as 15-35° C., and a reaction time of 1-100 seconds, such as 5-50seconds. The oxidation agent may be, for example, iodine (in someembodiments, provided as iodine water). In some embodiments, the molarratio of the oxidation agent to the phosphite ester group is 1:1 to100:1, preferably 5:1 to 50:1. In some embodiments, the oxidationreaction is performed in a mixed solvent in which the ratio oftetrahydrofuran:water:pyridine is 3:1:1-1:1:3.

In some embodiments, R₆ is B7 or B8:

wherein q₂ is as defined above.

In this case, the compound shown in the Formula (313) may be prepared bythe following preparation method comprising: contacting the compound asshown by Formula (314) with a compound as shown by Formula (A-1) or(A-2) in an organic solvent under amidation reaction condition in thepresence of an agent for amidation condensation and an organic base oftertiary amine, and isolating:

wherein the definitions and options of n1, n3, m1, m2, m3, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄, R₁₅, L₁, S₁, q2 and R_(k) are respectively as describedabove.

The amidation reaction condition may comprise a reaction temperature of0-100° C. and a reaction time of 1-48 hours. In some embodiments, theamidation reaction condition comprises a reaction temperature of 10-40°C. and a reaction time of 2-16 hours.

In some embodiments, the organic solvent is one or more of an alcoholsolvent, an epoxy solvent, an ether solvent, an haloalkane solvent,dimethyl sulfoxide, N,N-dimethylformamide, andN,N-diisopropylethylamine. In some embodiments, the alcohol solvent isone or more of methanol, ethanol and propanol, and in furtherembodiments, ethanol. In some embodiments, the epoxy solvent is dioxaneand/or tetrahydrofuran. In some embodiments, the ether solvent isdiethyl ether and/or methyl tertbutyl ether. In some embodiments, thehaloalkane solvent is one or more of dichloromethane, trichloromethaneand 1,2-dichloroethane. In some embodiments, the organic solvent isdichloromethane. The amount of the organic solvent is 3-50 L/mol, and infurther embodiments, 3-20 L/mol, with respect to the compound as shownby Formula (314).

In some embodiments, the agent for amidation condensation isbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate,3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one,4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride,2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) orO-benzotriazol-tetramethyluronium hexafluorophosphate, and in furtherembodiments, 3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one. Themolar ratio of the agent for amidation condensation to the compound asshown by Formula (314) may be 1:1 to 10:1, and in some embodiments,2.5:1 to 5:1.

In some embodiments, the organic base of tertiary amine is triethylamineor N,N-diisopropylethylamine, and in further embodiments,N,N-diisopropylethylamine. The molar ratio of the tertiary amine to thecompound as shown by Formula (314) may be 3:1 to 20:1, and in someembodiments, 5:1 to 10:1.

In some embodiments, the compounds of Formula (A-1) and (A-2) may beprepared by any suitable methods. For example, when R_(k) is a DMTrgroup, the compound of Formula (A-1) may be prepared by reacting calciumglycerate with DMTrCl. Similarly, the compound of Formula (A-2) may beprepared by contacting 3-amino-1,2-propanediol with a cyclic anhydrideand then reacting with DMTrCl, wherein the cyclic anhydride may have4-13 carbon atoms, and in some embodiments, 4-8 carbon atoms. Thoseskilled in the art would readily understand that the selections ofdifferent cyclic anhydrides correspond to different values for q₂ in thecompound of Formula (A-2). For example, when the cyclic anhydride issuccinic anhydride, q₂=1; when the cyclic anhydride is glutaricanhydride, q2=2, and so on.

In some variants, the compound of Formula (313) can also be prepared bysuccessively reacting the compound as shown by Formula (314) with thecyclic anhydride, 3-amino-1,2-propanediol, and DMTrCl. Those skilled inthe art would readily understand that these variants would not affectthe structure and function of the compound of Formula (313), and thesevariants can be readily achieved by those skilled in the art on thebasis of the above methods.

Similarly, the compound as shown by Formula (313) may be isolated fromthe reaction mixture by any suitable isolation methods. In someembodiments, the compound as shown by Formula (313) may be isolated byremoval of solvent via evaporation followed by chromatography, forexample, using the following two sets of chromatographic conditions forisolation: (1) normal phase purification: 200-300 mesh silica gelfiller, gradient elution of petroleum ether:ethylacetate:dichloromethane:N,N-dimethylformamide=1:1:1:0.5-1:1:1:0.6; and(2) reverse phase purification: C18 and C8 reverse phase fillers,gradient elution of methanol:acetonitrile=0.1:1-1:0.1. In someembodiments, the solvent may be directly removed to obtain a crudeproduct of the compound as shown by Formula (313), which may be directlyused in subsequent reactions.

In some embodiments, the compound as shown by Formula (314) may beprepared by the following preparation method comprising contacting thecompound as shown by Formula (315) with haloacetic acid in an organicsolvent under deprotection reaction condition, and then isolating:

wherein R₇ is selected from the groups as shown by Formula (330), (331),(332) and (333), and in some embodiments, R₇ has the structure as shownby Formula (330):

wherein the definitions and options of n1, n3, m1, m2, m3, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄, R₁₅, L₁ and S₁ are respectively as described above.

The haloacetic acid may be selected from one or more of dichloroaceticacid, trichloroacetic acid, monochloroacetic acid and trifluoroaceticacid, and in some embodiments, dichloroacetic acid.

The deprotection reaction condition may comprise a reaction temperatureof 0-100° C. and a reaction time of 0.1-24 hours, and in someembodiments comprises a reaction temperature of 10-40° C. and a reactiontime of 0.5-16 hours.

In some embodiments, the organic solvent is one or more of an epoxysolvent, an ether solvent, an haloalkane solvent, dimethyl sulfoxide,N,N-dimethylformamide, and N,N-diisopropylethylamine. In someembodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. Insome embodiments, the ether solvent is diethyl ether and/or methyltertbutyl ether. In some embodiments, the haloalkane solvent is one ormore of dichloromethane, trichloromethane and 1,2-dichloroethane. Insome embodiments, the organic solvent is dichloromethane. The amount ofthe organic solvent is 3-50 L/mol, and in further embodiments, 5-20L/mol, with respect to the compound as shown by Formula (315).

The molar ratio of the haloacetic acid to the compound as shown byFormula (315) may be 5:1 to 100:1, and in some embodiments, 10:1 to50:1.

Similarly, the compound as shown by Formula (314) may be isolated fromthe reaction mixture by any suitable isolation methods. In someembodiments, the compound as shown by Formula (314) may be isolated byremoval of solvent via evaporation followed by chromatography, forexample, using the following two sets of chromatographic conditions forisolation, (1) normal phase purification: 200-300 mesh silica gelfiller, gradient elution of dichloromethane:methanol=100:30-100:40; and(2) reverse phase purification: C18 and C8 reverse phase fillers,gradient elution of methanol:acetonitrile=0.1:1-1:0.1. In someembodiments, the solvent may be directly removed to obtain a crudeproduct of the compound as shown by Formula (314), which may be directlyused in subsequent reactions.

The compound as shown by Formula (315) may be prepared by the followingmethod comprising contacting the compound as shown by Formula (317) withthe compound as shown by Formula (316) in an organic solvent undercondensation reaction condition in the presence of an agent foramidation condensation and an organic base of tertiary amine, andisolating:

wherein the definitions and options of n1, n3, m1, m2, m3, R₇, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄, R₁₅, L₁ and S₁ are respectively as described above.

The compound of Formula (316) can be, such as, those disclosed in J. Am.Chem. Soc. 2014, 136, 16958-16961. Alternatively, the compounds ofFormula (316) may be prepared by those skilled in the art via variousmethods. For example, some compounds of Formula (316) may be preparedaccording to the methods as disclosed in Example 1 of U.S. Pat. No.8,106,022 B2, which is incorporated herein by reference in its entirety.

In some embodiments, the condensation reaction condition comprises areaction temperature of 0-100° C. and a reaction time of 0.1-24 hours.In some embodiments, the condensation reaction condition comprises areaction temperature is 10-40° C. and a reaction time is 0.5-16 hours.

The molar ratio of the compound as shown by Formula (316) to thecompound as shown by Formula (317) may be 2:1 to 10:1, and in someembodiments, 2.5:1 to 5:1.

In some embodiments, the organic solvent is one or more of acetonitrile,an epoxy solvent, an ether solvent, an haloalkane solvent, dimethylsulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In someembodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. Insome embodiments, the ether solvent is diethyl ether and/or methyltertbutyl ether. In some embodiments, the haloalkane solvent is one ormore of dichloromethane, trichloromethane and 1,2-dichloroethane. Insome embodiments, the organic solvent is acetonitrile. The amount of theorganic solvent may be 3-50 L/mol, and in some embodiments, 5-20 L/mol,with respect to the compound as shown by Formula (317).

In some embodiments, the agent for amidation condensation isbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate,3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one (DEPBT),O-benzotriazol-tetramethyluronium hexafluorophosphate or4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, and infurther embodiments, is 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinehydrochloride. The molar ratio of the agent for amidation condensationto the compound as shown by Formula (317) may be 2:1 to 10:1, and insome embodiments, is 2.5:1 to 5:1.

The organic base of tertiary amine may be N-methylmorpholine,triethylamine or N,N-diisopropylethylamine, and in some embodiments,N-methylmorpholine. The molar ratio of the tertiary amine to thecompound as shown by Formula (317) may be 3:1 to 20:1, and in someembodiments, is 5:1 to 10:1.

Similarly, the compound as shown by Formula (315) may be isolated fromthe reaction mixture by any suitable isolation methods. In someembodiments, the compound as shown by Formula (315) is isolated byremoval of solvent via evaporation followed by chromatography, forexample, using the following two sets of chromatographic conditions forisolation, (1) normal phase purification: 200-300 mesh silica gelfiller, gradient elution of dichloromethane:methanol=100:5-100:7; (2)reverse phase purification: C18 and C8 reverse phase fillers, gradientelution of methanol:acetonitrile=0.1:1-1:0.1. In some embodiments, thesolvent is directly removed to obtain a crude product of the compound asshown by Formula (315), which may be directly used in subsequentreactions.

In some embodiments, the compound of Formula (317) reacts with asufficient amount of one compound of Formula (316) in one batch toobtain the desired compound of Formula (315), wherein all S₁-L₁ moietiesare identical. In some embodiments, the compound of Formula (317) reactswith different compounds of Formula (316) in batches as desired, i.e.,the compounds of Formula (316) having different L₁ and/or S₁, so as toobtain the compound of Formula (315) having two or more types of S₁and/or L₁ therein. For example, 1 eq of the compound of Formula (317)may be firstly contacted with 2 eq of a first compound of Formula (316)to attach the first S₁-L₁ moieties to the two terminal primary aminegroups in the compound of Formula (317), and then contacted with the(n3+n1-1) eq of a second compound of Formula (316) to attach the secondS₁-L₁ moieties to the (n3+n1-1) secondary amine groups in the compoundof Formula (317), wherein the definitions and ranges of n3 and n1 are asdescribed above.

In some embodiments, the compound as shown by Formula (317) may beprepared by the following method comprising contacting the compound asshown by Formula (318) with aqueous methylamine solution underdeprotection reaction condition in the presence of an organic solvent,and isolating:

wherein the definitions and options of n1, n3, m1, m2, m3, R₇, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄ and R₁₅ are respectively as described above.

The deprotection reaction condition may comprise a reaction temperatureof 0-150° C. and a reaction time of 5-72 hours, and in some embodimentscomprises a reaction temperature of 20-80° C. and a reaction time of10-30 hours.

The organic solvent may be selected from alcohols, in some embodiments,is one of methanol, ethanol and isopropanol, and in some embodiments,methanol. The amount of the organic solvent may be 1-20 L/mol, and insome embodiments, is 1.5-10 L/mol, with respect to the compound as shownby Formula (318).

The concentration of the methylamine aqueous solution may be 30%-40% bymass, and the molar ratio of methylamine to the compound as shown byFormula (318) may be 10:1 to 500:1, and in some embodiments, 50:1 to200:1.

Similarly, the compound as shown by Formula (317) may be isolated fromthe reaction mixture using any suitable isolation methods. In someembodiments, the compound as shown by Formula (317) may be isolated byremoval of solvent via evaporation followed by chromatography, forexample, using the following two sets of chromatographic conditions forisolation, (1) normal phase purification: 200-300 mesh silica gelfiller, gradient elution of dichloromethane:methanol:aqueous ammonia (25wt %)=1:1:0.05-1:1:0.25; and (2) reverse phase purification: C18 and C8reverse phase fillers, gradient elution ofmethanol:acetonitrile=0.1:1-1:0.1. In some embodiments, the solvent maybe directly removed to obtain a crude product of the compound as shownby Formula (317), which may be directly used in subsequent reactions.

The compound as shown by Formula (318) may be prepared by the followingmethod comprising contacting the compound as shown by Formula (319) withtriphenylchloromethane (TrCl), diphenylethylphenylchloromethane,phenyldiethylphenylchloromethane or triethylphenylchloromethane (in someembodiments, with triphenylchloromethane (TrCl)) under substitutionreaction condition in the presence of an organic solvent, and isolating:

wherein the definitions and options of n1, n3, m1, m2, m3, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄ and R₁₅ are respectively as described above.

The substitution reaction condition may comprise a reaction temperatureof 0-100° C. and a reaction time of 5-72 hours, and in some embodimentscomprises a reaction temperature of 10-40° C. and a reaction time of10-30 hours.

Triphenylchloromethane (TrCl), diphenylethylphenylchloromethane,phenyldiethylphenylchloromethane or triethylphenylchloromethane arecommercially available. The molar ratio of triphenylchloromethane(TrCl), diphenylethylphenylchloromethane,phenyldiethylphenylchloromethane or triethylphenylchloromethane to thecompound as shown by Formula (319) may be 1:1 to 10:1, and in someembodiments, 1:1 to 3:1.

The organic solvent may be one or more of an epoxy solvent, an ethersolvent, an haloalkane solvent, dimethyl sulfoxide,N,N-dimethylformamide, and N,N-diisopropylethylamine. In someembodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. Insome embodiments, the ether solvent is diethyl ether and/or methyltertbutyl ether. In some embodiments, the haloalkane solvent is one ormore of dichloromethane, trichloromethane and 1,2-dichloroethane. Insome embodiments, the organic solvent is dichloromethane. The amount ofthe organic solvent may be 3-50 L/mol, and in some embodiments, 5-20L/mol, with respect to the compound as shown by Formula (319).

Similarly, the compound as shown by Formula (318) may be isolated fromthe reaction mixture by any suitable isolation methods. In someembodiments, the compound as shown by Formula (318) may be isolated byremoval of solvent via evaporation followed by chromatography, forexample, using the following two sets of chromatographic conditions forisolation, (1) normal phase purification: 200-300 mesh silica gelfiller, gradient elution of methanol:dichloromethane=0.01:1-0.5:1 orgradient elution of methanol:dichloromethane:ethyl acetate:petroleumether=0.1:1:1:1-1:1:1:1; and (2) reverse phase purification: C18 and C8reverse phase fillers, gradient elution ofmethanol:acetonitrile=0.1:1-1:0.1. In some embodiments, the solvent maybe directly removed to obtain a crude product of the compound as shownby Formula (318), which may be directly used in subsequent reactions.

In some embodiments, the compound as shown by Formula (319) may beprepared by the following method comprising contacting the compound asshown by Formula (320) with ethyl trifluoroacetate in an organic solventunder substitution reaction condition, and isolating:

wherein the definitions and options of n1, n3, m1, m2, m3, R₁₀, R₁₁,R₁₂, R₁₃, R₁₄ and R₁₅ are respectively as described above.

In some embodiments, the organic solvent is one or more of acetonitrile,an epoxy solvent, an ether solvent, an haloalkane solvent, dimethylsulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In someembodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. Insome embodiments, the ether solvent is diethyl ether and/or methyltertbutyl ether. In some embodiments, the haloalkane solvent is one ormore of dichloromethane, trichloromethane and 1,2-dichloroethane. Insome embodiments, the organic solvent is acetonitrile. The amount of theorganic solvent may be 1-50 L/mol, and in some embodiments, 1-20 L/mol,with respect to the compound as shown by Formula (320).

The substitution reaction condition may comprise a reaction temperatureof 0-100° C. and a reaction time of 5-72 hours, and in some embodimentscomprises a reaction temperature of 10-40° C. and a reaction time of10-30 hours.

The compound as shown by Formula (320) may be commercially available, orobtained by those skilled in the art via the known methods. For example,in the case that m1=m2=m3=3, n1=1, n3=2, and R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ andR₁₅ are all H, the compound as shown by Formula (320) is commerciallyavailable from Alfa Aesar Inc.

The molar ratio of ethyl trifluoroacetate to the compound as shown byFormula (320) may be 2:1 to 10:1, and in some embodiments, 3:1 to 5:1.

Similarly, the compound as shown by Formula (319) may be isolated fromthe reaction mixture using any suitable isolation methods. In someembodiments, the compound as shown by Formula (319) may be isolated byremoval of solvent via evaporation followed by chromatography, forexample, using the following two sets of chromatographic conditions forisolation: (1) normal phase purification: 200-300 mesh silica gelfiller, gradient elution of methanol:dichloromethane=0.01:1-0.5:1 orgradient elution of methanol:dichloromethane:ethyl acetate:petroleumether=0.1:1:1:1-1:1:1:1; and (2) reverse phase purification: C18 and C8reverse phase fillers, gradient elution ofmethanol:acetonitrile=0.1:1-1:0.1. In some embodiments, the solvent maybe directly removed to obtain a crude product of the compound as shownby Formula (319), which may be directly used in subsequent reactions.

The oligonucleotide conjugate of the present disclosure may also be usedin combination with other pharmaceutically acceptable excipients, whichmay be one or more of the various conventional formulations or compoundsin the art. For details, please refer to the above description of thepharmaceutical compositions of the present disclosure.

Use of the Double-Stranded Oligonucleotide, Pharmaceutical Compositionand Oligonucleotide Conjugate of the Present Disclosure

In some embodiments, the present disclosure provides use of thedouble-stranded oligonucleotide, pharmaceutical composition and/oroligonucleotide conjugate of the present disclosure in the manufactureof a medicament for treating and/or preventing pathological conditionsor diseases caused by the expression of specific genes in cells. In someembodiments, the specific gene may be a gene abnormally expressed inhepatocytes. In some embodiments, the specific gene may be an endogenousgene expressed in liver. In some embodiments, the specific gene may bethe gene of a pathogen reproduced in liver. In some embodiments, thespecific gene is selected from the gene ApoB, ApoC, ANGPTL3, PCSK9,SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV and HCV. In someembodiments, the specific gene is selected from the gene of hepatitis Bvirus, the gene of angiopoietin-like protein 3, and the gene ofapolipoprotein C3. Correspondingly, the diseases are selected fromchronic liver disease, hepatitis, hepatic fibrosis, liver proliferativediseases and dyslipidemia. In some embodiments, the dyslipidemia ishypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

In some embodiments, the present disclosure provides a method fortreating pathological conditions or diseases caused by abnormalexpression of specific genes, comprising administering an effectiveamount of the double-stranded oligonucleotides, pharmaceuticalcompositions and/or oligonucleotide conjugates of the present disclosureto a subject in need thereof. In some embodiments, the specific gene isselected from the gene ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1,FVII, STAT3, p53, HBV and HCV. In some embodiments, the specific gene isselected from the gene of hepatitis B virus, the gene ofangiopoietin-like protein 3, and the gene of apolipoprotein C3.Correspondingly, the diseases are selected from chronic liver disease,hepatitis, hepatic fibrosis, liver proliferative diseases anddyslipidemia. In some embodiments, the dyslipidemia ishypercholesterolemia, hypertriglyceridemia or atherosclerosis. In someembodiments, the conjugate provided by the present disclosure may alsobe used to treat other liver diseases, including diseases characterizedby undesired cell proliferation, blood diseases, metabolic diseases, anddiseases characterized by inflammation. Proliferative diseases of livermay be benign or malignant diseases such as cancer, hepatocellularcarcinoma (HCC), hepatic metastasis or hepatoblastoma. Liver hematologyor inflammatory diseases may be diseases that involve coagulationfactors, complement-mediated inflammation or fibrosis. Liver metabolicdiseases include dyslipidemia and irregular glucose regulation. In someembodiments, the method comprises administering one or moredouble-stranded oligonucleotides having a high degree of homology to thegene sequences involved in the diseases.

In some embodiments, the present disclosure provides a method forinhibiting the expression of specific genes in cells, comprisingcontacting an effective amount of the double-stranded oligonucleotide,pharmaceutical composition and/or oligonucleotide conjugate of thepresent disclosure with the cells.

The purpose of preventing and/or treating pathological conditions ordiseases caused by the expression of specific genes in cells may beachieved through the mechanism of regulating gene expression byadministering the double-stranded oligonucleotide, pharmaceuticalcomposition and/or oligonucleotide conjugate of the present disclosureto a subject in need thereof. Therefore, the double-strandedoligonucleotide, pharmaceutical composition and/or oligonucleotideconjugate of the present disclosure may be used for preventing and/ortreating the pathological conditions or diseases of the presentdisclosure, or for preparing a medicament for preventing and/or treatingthe pathological conditions or diseases of the present disclosure.

As used herein, the term “administration/administer” refers to thedelivery of the double-stranded oligonucleotide, pharmaceuticalcomposition and/or oligonucleotide conjugate into a subject's body by amethod or a route that at least partly locates the double-strandedoligonucleotide, pharmaceutical composition and/or oligonucleotideconjugate at a desired site to produce a desired effect. Suitableadministration routes for the methods of the present disclosure includetopical administration and systemic administration. In general, topicaladministration results in the delivery of more double-strandedoligonucleotides, pharmaceutical compositions and/or oligonucleotideconjugates to a particular site compared wth the whole body of thesubject; whereas systemic administration results in the delivery of thedouble-stranded oligonucleotides, pharmaceutical compositions and/oroligonucleotide conjugates to substantially the whole body of thesubject. Considering that the present disclosure aims to provide a meansfor preventing and/or treating pathological conditions or diseasescaused by the expression of specific genes in hepatocytes, in someembodiments, an administration mode capable of delivering drugs to liveris used.

The administration to a subject may be achieved by any suitable routesknown in the art, including but not limited to, oral or parenteralroute, such as intravenous administration, intramuscular administration,subcutaneous administration, transdermal administration, intratrachealadministration (aerosol), pulmonary administration, nasaladministration, rectal administration and topical administration(including buccal administration and sublingual administration). Theadministration frequency may be once or more times daily, weekly,biweekly, triweekly, monthly, or yearly.

The dose of the double-stranded oligonucleotide, pharmaceuticalcomposition and/or oligonucleotide conjugate of the present disclosuremay be a conventional dose in the art, which may be determined accordingto various parameters, especially age, weight and gender of a subject.Toxicity and efficacy may be measured in cell cultures or experimentalanimals by standard pharmaceutical procedures, for example, bydetermining LD50 (the lethal dose that causes 50% population death) andED50 (the dose that can cause 50% of the maximum response intensity in aquantitative response, and that causes 50% of the experimental subjectsto have a positive response in a qualitative response). The dose rangefor human may be derived based on the data obtained from cell cultureassays and animal studies.

When administering the double-stranded oligonucleotide, pharmaceuticalcomposition and/or oligonucleotide conjugate of the present disclosure,for example, to male or female C57BL/6J or C3H/HeNCrlVr mice of 6-12weeks old and 18-25 g body weight, for an oligonucleotide conjugateformed by a double-stranded oligonucleotide and pharmaceuticallyacceptable conjugating molecules, the amount of the double-strandedoligonucleotide may be 0.001-100 mg/kg body weight, in some embodiments0.01-50 mg/kg body weight, in further embodiments 0.05-20 mg/kg bodyweight, in still further embodiments 0.1-15 mg/kg body weight, and instill yet further embodiments 0.1-10 mg/kg body weight, as calculatedbased on the amount of the double-stranded oligonucleotide in thedouble-stranded oligonucleotide, pharmaceutical composition and/oroligonucleotide conjugate. When administering the double-strandedoligonucleotide, pharmaceutical composition and/or oligonucleotideconjugate of the present disclosure, the above amounts are preferred.

In addition, the purpose of inhibiting the expression of specific genesin hepatocytes may also be achieved through the mechanism of regulatinggene expression by introducing the double-stranded oligonucleotide,pharmaceutical composition and/or oligonucleotide conjugate of thepresent disclosure into the hepatocytes with abnormal expression of thespecific genes. In some embodiments, the hepatocytes are hepatitiscells, and in some embodiments are HepG2.2.15 cells. In someembodiments, the hepatocytes may be selected from hepatoma cell linessuch as Hep3B, HepG2 or Huh7, and isolated liver primary cells, and insome embodiments are Huh7 hepatoma cells.

In the case where the expression of specific genes in hepatocytes isinhibited by using the method of the present disclosure, the amount ofthe double-stranded oligonucleotide in the provided double-strandedoligonucleotide, pharmaceutical composition and/or oligonucleotideconjugate can be readily determined by those skilled in the artaccording to the desired effects. For example, in some embodiments wherethe double-stranded oligonucleotide, pharmaceutical composition and/oroligonucleotide conjugate is an siRNA conjugate, the amount of siRNA inthe siRNA conjugate provided is an amount sufficient to reduce theexpression of the target gene and result in an extracellularconcentration of 1 pM to 1 μM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nMor 0.05 nM to about 5 nM on the surface of the target cells. The amountrequired to achieve this local concentration will vary with variousfactors, including the delivery method, the delivery site, the number ofcell layers between the delivery site and the target cells or tissues,the delivery route (topical or systemic), etc. The concentration at thedelivery site may be significantly higher than that on the surface ofthe target cells or tissues.

Kit

In another aspect, the present disclosure provides a kit comprising theabove double-stranded oligonucleotide, the above pharmaceuticalcomposition and/or the above oligonucleotide conjugate.

In some embodiments, the kit of the present disclosure provides thedouble-stranded oligonucleotide in one container. In some embodiments,the kit of the present disclosure comprises a container comprisingpharmaceutically acceptable excipients. In some embodiments, the kis ofthe present disclosure further comprises additional ingredients, such asstabilizers or preservatives. In some embodiments, the kit comprises atleast one additional therapeutic agent in other container than thecontainer comprising the double-stranded oligonucleotide of the presentdisclosure. In some embodiments, the kit comprises an instruction formixing the double-stranded oligonucleotide with pharmaceuticallyacceptable carriers and/or adjuvants or other ingredients (if any).

In a kit of the present disclosure, the double-stranded oligonucleotidesand the pharmaceutically acceptable carriers and/or adjuvants, thecompositions and/or conjugates comprising said double-strandedoligonucleotides, and/or the pharmaceutically acceptable adjuvants maybe provided in any form, e.g., in a liquid form, a dry form, or alyophilized form. In some embodiments, the double-strandedoligonucleotides and the pharmaceutically acceptable carriers and/oradjuvants, the compositions and/or conjugates comprising saiddouble-stranded oligonucleotides, and optional pharmaceuticallyacceptable adjuvants are substantially pure and/or sterile. In someembodiments, sterile water may be provided in the kit of the presentdisclosure.

Hereinafter, the present disclosure will be further illustrated withreference to the examples, but is not limited thereto.

Without wishing to be limited, the invention is described in furtherdetails in the following embodiments and the Examples regarding theexemplary embodiments where the double-stranded oligonucleotide in thecompositions and/or oligonucleotide conjugates of the present disclosureis a small interfering RNA (siRNA). In this case, the double-strandedoligonucleotide, composition, and oligonucleotide conjugate of thepresent disclosure are an siRNA, a composition comprising an siRNA andan siRNA conjugate, respectively. In the context of the presentdisclosure, the siRNA, the compositions comprising siRNA and the siRNAconjugates in these embodiments are also referred to as the siRNA, thesiRN compositions and the siRNA conjugates of the present disclosurejust for convenience of description. It does not mean that thedouble-stranded oligonucleotide of the present disclosure can only besiRNA, instead, the double-stranded oligonucleotide may be othervariants disclosed in the present disclosure or known to those skilledin the art, such as small activating RNA (saRNA). It can be envisagedthat, based on the detailed illustration of the siRNA, the compostionscomprising siRNA, and the siRNA conjugates, other functionaldouble-stranded oligonucleotides would work similarly alone, or whenforming the compositions and/or conjugates of the present disclosure.

Advantageous Effects

In some embodiments, the double-stranded oligonucleotide, composition oroligonucleotide conjugate of the present disclosure can have higherstability, lower toxicity, and/or higher activity in vivo. In someembodiments, the double-stranded oligonucleotide of the presentdisclosure is saRNA. In some embodiments, the saRNA, saRNA compositionor saRNA conjugate of the present disclosure exhibits an improvementpercentage of target gene expression of at least 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% in vivo. In some embodiments, thedouble-stranded oligonucleotide of the present disclosure is siRNA. Insome embodiments, the siRNA, siRNA composition or siRNA conjugate of thepresent disclosure exhibits an inhibition percentage of target geneexpression of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% invivo. In some embodiments, the siRNA, siRNA composition or siRNAconjugate of the present disclosure exhibits an inhibition percentage ofHBV gene expression of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 95% in vivo. In some embodiments, the siRNA, siRNA composition orsiRNA conjugate of the present disclosure exhibits an inhibitionpercentage of HBV gene expression in liver of at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% in vivo. In some embodiments, the siRNA,siRNA composition or siRNA conjugate of the present disclosure exhibitsan inhibition percentage of HBV gene expression in liver in animalmodels of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% invivo. In some embodiments, the siRNA, siRNA composition or siRNAconjugate of the present disclosure exhibits an inhibition percentage ofHBV surface antigen expression of at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 95% in vivo. In some embodiments, the siRNA, siRNAcomposition or siRNA conjugate of the present disclosure exhibits aninhibition percentage of ANGPTL3 gene expression of at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95% in vivo. In some embodiments, thesiRNA, siRNA composition or siRNA conjugate of the present disclosureexhibits an inhibition percentage of ANGPTL3 gene expression in liver ofat least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in vivo. In someembodiments, the siRNA, siRNA composition or siRNA conjugate of thepresent disclosure exhibits an inhibition percentage of ANGPTL3 geneexpression in liver in animal models of at least 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% in vivo. In some embodiments, the siRNA,siRNA composition or siRNA conjugate of the present disclosure exhibitsan inhibition percentage of ANGPTL3 gene expression in liver in humansubjects of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% invivo. In some embodiments, the siRNA, siRNA composition or siRNAconjugate of the present disclosure exhibits an inhibition percentage ofApoC3 gene expression of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% in vivo. In some embodiments, the siRNA, siRNA compositionor siRNA conjugate of the present disclosure exhibits an inhibitionpercentage of ApoC3 gene expression in liver of at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95% in vivo. In some embodiments, the siRNA,siRNA composition or siRNA conjugate of the present disclosure exhibitan inhibition percentage of ApoC3 gene expression in liver in animalmodels of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% invivo. In some embodiments, the siRNA, siRNA composition or siRNAconjugate of the present disclosure exhibit an inhibition percentage ofApoC3 gene expression in liver in human subjects of at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95% in vivo. In some embodiments, thedouble-stranded oligonucleotide, composition or oligonucleotideconjugate of the present disclosure exhibits no significant off-targeteffect. An off-target effect may be for example inhibition of normalexpression of a gene which is not the target gene. It is considered thatif the binding/inhibition of the expression of an off-target gene is50%, 40%, 30%, 20%, or 10% lower than that of the target activity, thenthe off-target effect is not significant.

According to some embodiments of the present disclosure, the siRNA,siRNA composition or siRNA conjugate of the present disclosure exhibitsan excellent inhibitory effect. For example, according to one embodimentof the present disclosure, the siRNA conjugate of the present disclosureexhibits excellent property of inhibiting HBV gene expression: aninhibition percentage of 66.9-90.9% of HBV gene expression in the liverof HBV model mice at a dose of 1 mg/kg, along with a low off-targeteffect. Meanwhile, the siRNA conjugate of the present disclosure furthereffectively reduces the expression of HBV surface antigen and HBV DNA inHBV model mice. In particular, compared with those provided in the priorart, the specific siRNA conjugate formed by the specifically modifiedsiRNA and the specific conjugating molecule provided by the presentdisclosure, exhibits an consistent and excellent inhibitory effect onHBV expression at low doses over a period of up to 140 days.

According to one embodiment of the present disclosure, the siRNAconjugate of the present disclosure exhibits excellent property ofinhibiting HBV gene expression: an inhibition percentage of 81.7-89.2%of HBV gene expression in the liver of HBV model mice at a dose of 1mg/kg, along with a low off-target effect. Meanwhile, the siRNAconjugate of the present disclosure further effectively reduces theexpression of HBV surface antigen and HBV DNA in HBV model mice. Inparticular, compared with the conjugate formed from the conjugatingmolecule provided in the prior art, the specific siRNA conjugate formedby the specifically modified siRNA and the specific conjugating moleculeprovided by the present disclosure, exhibits an consistent and excellentinhibitory effect on HBV expression at low doses over a period of up to84 days.

According to one embodiment of the present disclosure, the siRNAconjugate of the present disclosure exhibits excellent property ofinhibiting HBV gene expression: an inhibition percentage of up to 93.8%of HBV gene expression in the liver of HBV model mice at a dose of 1mg/kg, along with a low off-target effect. Meanwhile, the siRNAconjugate of the present disclosure further effectively reduces theexpression of HBV surface antigen in HBV model mice, achieves aninhibition percentage of 90% or higher for HBV surface antigenexpression even at a dose of 3 mg/kg, and effectively inhibits HBV DNA.In particular, compared with reference conjugates, the specific siRNAconjugate formed by the specifically modified siRNA and the specificconjugating molecule provided by the present disclosure, exhibits anconsistent and higher inhibitory effect on HBV expression at lower dosesover a period of up to 21 days.

According to one embodiment of the present disclosure, the siRNAconjugate of the present disclosure exhibits excellent property ofinhibiting HBV gene expression: an inhibition percentage of up to 93.63%of gene expression for HBV X gene region in the liver of HBV model miceat a dose of 1 mg/kg, along with a low off-target effect. Meanwhile, thesiRNA conjugate of the present disclosure further effectively reducesthe expression of HBV surface antigen in HBV model mice, achieves aninhibition percentage of 95% or higher for HBV surface antigenexpression even at a dose of 3 mg/kg, and effectively inhibits HBV DNA.In particular, compared with the conjugate formed from the conjugatingmolecule provided in the prior art, the specific siRNA conjugate formedby the specifically modified siRNA and the specific conjugating moleculeprovided by the present disclosure, exhibits an consistent and excellentinhibitory effect on HBV expression at lower doses over a period of upto 56 days and an inhibition percentage of 90% or higher for HBV X mRNA.

In some embodiment, the siRNA conjugates of the present disclosure canexhibit an excellent efficiency of inhibiting ANGPTL3 mRNA andsignificantly down-regulate blood lipid level. For example, in someembodiments, an inhibition percentage of up to 95% or higher for ANGPTL3mRNA in mice is achieved on day 14 after single subcutaneousadministration; in some embodiments, the maximum inhibition percentageof triglyceride (TG) is 93% and the maximum inhibition percentage oftotal cholesterol (CHO) is 83% after single subcutaneous administration;and the inhibition percentage of TG is maintained at 55% or higher andthe inhibition percentage of CHO is maintained at 40% or higher on day154 after administration. In particular, compared with the conjugateformed from the conjugating molecule provided in the prior art, thesiRNA conjugate of the present disclosure exhibits more excellentinhibition percentage of gene expression and enhanced capability ofreducing blood lipid; and the siRNA conjugate of the present disclosureexhibits consistent and excellent hypolipidemic effects at low doses andlow administration frequency over a period of up to 189 days.

In some embodiments, the siRNA conjugates of the present disclosureexhibits excellent property of inhibiting ApoC3 gene expression: aninhibition percentage of at least 88% of ApoC3 gene expression in theliver of high-lipid model mice at a dose of 1 mg/kg. In particular,compared with the conjugate formed from the conjugating moleculeprovided in the prior art, the modified siRNA and siRNA conjugateprovided by the present disclosure exhibit excellent inhibitionpercentage of gene expression and low off-target effect; and the siRNAconjugate of the present disclosure exhibits consistent and excellenthypolipidemic effects at low doses and low administration frequency overa period of up to 189 days.

In some embodiments, the siRNA conjugates of the present disclosureexhibit low toxicity and good safety in animal models. For example, insome embodiments, no obviously toxic response is observed even when theconjugate of the present disclosure is administered to C57BL/6J mice ata concentration of 100-fold higher than the minimal effectiveconcentration (calculated based on the minimal effective concentrationof 3 mg/kg).

The above instances indicate that the siRNA, siRNA composition and siRNAconjugate of the present disclosure can effectively reduce geneexpression in the target cell and exhibit excellent delivery potency.

EXAMPLES

Hereinafter, the present disclosure will be described in detail withreference to the examples. Unless otherwise specified, the agents andculture media used in following examples are all commercially available,and the procedures used such as nucleic acid electrophoresis andreal-time PCR are all performed according to methods described inMolecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

HEK293A cells were provided by Nucleic acid technology laboratory,Institute of Molecular Medicine, Peking University and cultured in DMEMcomplete media (Hyclone company) containing 20% fetal bovine serum (FBS,Hyclone company), 0.2 v % Penicillin-Streptomycin (Gibco, Invitrogencompany) at 37° C. in an incubator containing 5% CO₂/95% air.

HepG2.2.15 cells were purchased from ATCC and cultured in DMEM completemedium (Gibco) containing 10% fetal bovine serum (FBS, Gibco), 2 mML-glutamine (Gibco) and 380 μg/ml G418 at 37° C. in an incubatorcontaining 5% CO₂/95% air.

Huh7 cells were purchased from ATCC and cultured in DMEM complete medium(Gibco) containing 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine(Gibco) and 380 μg/ml G418 at 37° C. in an incubator containing 5%CO₂/95% air.

Unless otherwise specified, Lipofectamine™ 2000 (Invitrogen company) wasused as a transfection agent when cells were transfected with varioussynthesized siRNA or siRNA conjugates below. Detailed procedures wasperformed with reference to the instruction provided by manufacturer.

Unless otherwise specified, ratios of the agents provided below are allcalculated by volume ratio (v/v).

The animal models used are as follows:

C57BL/6N mice: 6-8 weeks old, purchased from Beijing Vital RiverLaboratory Animal Technology Co., Ltd., hereinafter referred to as C57mice;

SD rats: provided by Beijing Vital River Laboratory Animal TechnologyCo., Ltd.;

HBV transgenic mice: C57BL/6-HBV, Strain name: B6-Tg HBV/Vst (1.28 copy,genotype A), purchased from Beijing Vital River Laboratory AnimalTechnology Co., Ltd. Mice with COI>10⁴ (hereinafter referred as 1.28copy mice) are selected before experiments;

HBV transgenic mice C57BL/6J-Tg (Alb1HBV) 44Bri/J: purchased fromDepartment of Laboratory Animal Science, Health Science Center, PekingUniversity;

HBV transgenic mice M-TgHBV, purchased from Department of Animal,Shanghai Public Health Center. The preparation methods of transgenicmice are described in Ren J. et al., in J. Medical Virology. 2006,78:551-560;

AAV-HBV transgenic mice: AAV-HBV model prepared according to the methodin the (Xiaoyan Dong et al., Chin J Biotech 2010, May 25; 26(5):679-686) by using rAAV8-1.3HBV, D type (ayw) virus (purchased fromBeijing FivePlus Molecular Medicine Institute Co. Ltd., 1×10¹² viralgenome (v.g.)/mL, Lot No. 2016123011). The rAAV8-1.3HBV was diluted to5×10¹¹ v.g./mL with sterile PBS before experiments. 200 μL of thediluted rAAV8-1.3HBV was injected into each mouse, i.e., 1×10¹¹ v.g. permouse. The orbital blood (about 100 μL) was taken from each mouse on day28 after virus injection to collect serum for detection of HBsAg and HBVDNA;

Low concentration AAV-HBV transgenic mice: prepared by usingsubstantially the same modeling method as above, except that the viruswas diluted to 1×10¹¹ v.g./mL with sterile PBS before experiments. 100μL virus was injected into each mouse, i.e., 1×10¹⁰ v.g. per mouse;

BALB/c mice: 6-8 weeks old, purchased from Beijing Vital RiverLaboratory Animal Technology Co., Ltd.;

ob/ob mice: 6-8 weeks old, purchased from Changzhou Cavens LaboratoryAnimal Co., Ltd.;

Human APOC3 transgenic mice: B6; CBA-Tg(APOC3)3707Bres/J, purchased fromJackson Laboratory, USA;

Metabolic syndrome monkey: provided by Non-human Primate ResearchCenter, Institute of Molecular Medicine, Peking University.

Unless otherwise specified, the following data of effect experiments inintro in vivo are expressed as X±SEM, and the data are analyzed withstatistical analysis software Graphpad prism 5.0. The data are initiallytested for normal distribution and homogeneity of variance. If the datameet normal distribution (p>0.20) and homogeneity of variance (p>0.10),then comparison among groups would be performed by LSD method usingsingle-factor analysis of variance for multiple comparisons. P<0.05 isconsidered as being statistically significant. If the data fail to meetnormal distribution and homogeneity of variance, comparison among groupswould be performed by Krushkal-Wallis H method for Non-parametric Test.If the results obtained by Krushkal-Wallis H test are statisticallysignificant (p<0.05), pairwise comparisons among groups would beconducted after rank transformation of the data. P<0.05 is considered asbeing statistically significant.

Preparation Example 1 Preparation of siRNA of the Present Disclosure

In this preparation Example, siRNAs listed in Table 2 were synthesizedaccording to the following method.

TABLE 2 Synthesized siRNAs SEQ ID siRNA No. Sequence Direction: 5′-3′ NOsiRNA1 siAN1M3SVP S CmsCmsAmAmGmAmGfCfAfCmCmAmAmG 137 mAmAmCmUmAm ASVP-UmsAfsGmUmUmCfUmUmGmGmUmGm 138 CmUfCmUfUmGmGmsCmsUm siRNA2 siAN1M3SPS CmsCmsAmAmGmAmGfCfAfCmCmAmAmG 139 mAmAmCmUmAm ASP-UmsAfsGmUmUmCfUmUmGmGmUmGmC 140 mUfCmUfUmGmGmsCmsUm siRNA3 siAN1M3SPsS CmsCmsAmAmGmAmGfCfAfCmCmAmAmG 141 mAmAmCmUmAm ASPs-UmsAfsGmUmUmCfUmUmGmGmUmGmC 142 mUfCmUfUmGmGmsCmsUm siRNA4 siAN1M3S SCmsCmsAmAmGmAmGfCfAfCmCmAmAmG 143 mAmAmCmUmAm ASUmsAfsGmUmUmCfUmUmGmGmUmGmCm 144 UfCmUfUmGmGmsCmsUm Comparative siHBa1M5S CmCmUmUmGAGGCmAUmACmUmUmCmA 145 siRNA1 AAdTsdT ASUfUmUfGAAGUfAUGCCUfCAAGGdTsdT 146 Comparative siHBa1 SCGUGUGCACUUCGCUUCAA 147 siRNA2 AS UUGAAGCGAAGUGCACACGGU 148 ComparativeANG S CGUGUGCACUUCGCUUCAA 149 siRNA3 AS UUGAAGCGAAGUGCACACGGU 150Comparative 65695 S GUGUGCACUUCGCUUCACA 151 siRNA4 ASUGUGAAGCGAAGUGCACACUU 152 *S: sense strand; AS: antisense strand Note:C, G, U, and A represents the base components of the nucleotides; dTrepresents a deoxythymine nucleotide; m represents that the nucleotideadjacent to the left side of the letter m is a 2′-methoxy modifiednucleotide; f represents that the nucleotide adjacent to the left sideof the letter f is a 2′-fluoro modified nucleotide; s represents thatthe two nucleotides adjacent to both sides of the letter s are linked bya phosphorothioate linkage; VP represents that the nucleotide adjacentto the right side of the letter VP is a vinyl phosphate modifiednucleotide; P represents that the nucleotide adjacent to the right sideof the letter P is a phosphate nucleotide; Ps represents that thenucleotide adjacent to the right side of the letters Ps is aphosphorothioate modified nucleotide.

(1-1) Synthesis of a Sense Strand of siRNA

Nucleoside monomers are linked one by one in 3′ to 5′ directionaccording to the above sequences by starting the cycles using auniversal solid phase support (UnyLinker™ loaded NittoPhase® HL SolidSupports, Kinovate Life Sciences Inc.). The linking of each nucleosidemonomer included a four-step reaction of deprotection, coupling,capping, and oxidation. The synthesis conditions are as follows:

The nucleoside monomers are provided in a 0.1 M acetonitrile solution.The condition for deprotection reaction in each step is identical, i.e.,a temperature of 25° C., a reaction time of 70 seconds, a solution ofdichloroacetic acid in dichloromethane (3% v/v) as a deprotection agent,and a molar ratio of dichloroacetic acid to the protecting group on thesolid phase support of 4,4′-dimethoxytrityl of 5:1.

The condition for coupling reaction in each step is identical, includinga temperature of 25° C., a molar ratio of the nucleic acid sequencelinked to the solid phase support to nucleoside monomers of 1:10, amolar ratio of the nucleic acid sequence linked to the solid phasesupport to a coupling agent of 1:65, a reaction time of 600 seconds, and0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole as a couplingagent.

The condition for capping reaction in each step is identical, includinga temperature of 25° C. and a reaction time of 15 seconds, a mixedsolution of Cap 1 and Cap 2 in a molar ratio of 1:1 as a capping agent,and a molar ratio of the capping agent to the nucleic acid sequencelinked to the solid phase support of 1:1:1 (anhydride:N-methylimidazole:the nucleic acid sequence linked to the solid phase support).

The condition for oxidation reaction in each step is identical,including a temperature of 25° C., a reaction time of 15 seconds, and0.05 M iodine water as an oxidation agent; and a molar ratio of iodineto the nucleic acid sequence linked to the solid phase support in thecoupling step of 30:1. The reaction is carried out in a mixed solvent inwhich the ratio of tetrahydrofuran:water:pyridine is 3:1:1.

When two nucleotides in the target sequence is linked via aphosphorothioate linkage, the following sulfurization reaction step isused to replace the oxidation reaction step during linking of the laterof the two nucleotides: the condition for sulfurization reaction in eachstep is identical, including a temperature of 25° C., a reaction time of300 seconds, and xanthane hydride as a sulfurization agent; a molarratio of the sulfurization agent to the nucleic acid sequence linked tothe solid phase support in the coupling step of 120:1. The reaction iscarried out in a mixed solvent in which the ratio ofacetonitrile:pyridine is 1:1.

The conditions for cleavage and deprotection are as follows: adding thesynthesized nucleotide sequence linked to the support into 25 wt %aqueous ammonia to react for 16 hours at 55° C., and the aqueous ammoniais in an amount of 0.5 ml/μmol. The liquid is removed, and the residueis concentrated in vacuum to dryness. After treatment with aqueousammonia, the product is dissolved in 0.4 ml/μmol N-methylpyrrolidone,and then added with 0.3 ml/μmol triethylamine and 0.6 ml/μmoltriethylamine trihydrofluoride, with respect to the amount of the singlestrand nucleic acid, for removing the 2′-TBDMS protection on ribose.When 2′-positions of all nucleotides in the target sequence weremodified hydroxyl groups, the step of removing the 2′-TBDMS protectionon ribose is not included in the conditions for cleavage anddeprotection.

Purification and desalination: purification of the nucleic acid isachieved by using a preparative ion chromatography column (Source 15Q)with a gradient elution of NaCl. Specifically, eluent A: 20 mM sodiumphosphate (pH 8.1), solvent: water/acetonitrile=9:1 (v/v); eluent B: 1.5M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent:water/acetonitrile=9:1 (v/v); elution gradient: eluent A:eluentB=100:0-50:50. The eluate is collected, combined and desalted by using areverse phase chromatography column. The specific conditions includeusing a Sephadex column (filler: Sephadex-G25) for desalination anddeionized water for eluting.

Detection: the purity is determined by ion exchange chromatography(IEX-HPLC); and the molecular weight is analyzed by liquidchromatography-mass spectrometry (LC-MS), and compared with thecalculated value.

Thus, a sense strand S of siRNA is synthesized in the above step.

(1-2) Synthesis of an Antisense Strand

In this step, an antisense strand AS of siRNA is synthesized by using auniversal solid phase support (UnyLinker™ loaded NittoPhase® HL SolidSupports, Kinovate Life Sciences Inc.) under the same conditions as thatin the synthesis of the sense strand, including conditions ofdeprotection, coupling, capping, oxidation and/or sulfurizationreaction, deprotection, cleavage, and isolation in the solid phasesynthesis method.

Therein, the vinyl phosphate and 2′-methoxy modified uridine monomer(VP-Um) is synthesized according to the following method:

(1-2-1) Synthesis of VP-U-2

A VP-U-2 molecule is synthesized according to the following method:

A 2′-methoxy modified uracil nucleoside (2′-OMe-U, 51.30 g, 91.6 mmol),tertbutyl diphenylchlorosilane (TBDPSCl, 50.35 g, 183.2 mmol), andimidazole (12.47 g, 183.2 mmol) were mixed and dissolved in 450 ml ofN,N-dimethylformamide (DMF) to react under stirring at room temperaturefor 20 hours. DMF was removed by evaporation, and the residue wasdissolved in 600 ml of dichloromethane and washed with 300 ml ofsaturated sodium bicarbonate. The aqueous phase isolated was extractedthree times, each with 300 ml of dichloromethane. All organic phaseswere combined, washed with 5% oxalic acid until the pH of the aqueousphase is <5. The solvent was evaporated to dryness to give a crudeproduct of VP-U-1, which was directly used in the subsequent synthesisof VP-U-2.

The crude product VP-U-1 was dissolved in 100 ml of dichloromethane, andthen stirred for 10 minutes in an ice bath. 450 ml of 2%p-toluenesulfonic acid solution (with a mixed solvent of methanol anddichloromethane in a volume ratio of 3:7) pre-cooled in a refrigeratorat 4° C. was added to react for 10 minutes. The reaction was quenched byaddition of 200 ml of saturated sodium bicarbonate. The organic phaseobtained was washed by addition of saturated sodium bicarbonate solutionto pH=8. Aqueous phases were combined and extracted twice, each with 200ml of dichloromethane. All organic phases were combined and washed oncewith 200 ml of saturated brine. The solvent was removed by evaporation,and the residue was purified by using a normal phase silica gel column(200-300 mesh). The column was packed with petroleum ether and elutedwith a gradient elution of petroleum ether:ethylacetate:dichloromethane:methanol=1:1:1:0.05-1:1:1:0.25. The eluate wascollected, the solvent was removed by evaporation under reducedpressure, and the residue was foam-dried in a vacuum oil pump to give atotal of 40.00 g of pure product VP-U-2. 1H NMR (400 MHz, DMSO-d6) δ7.96 (d, J=7.8 Hz, 1H), 7.64 (dtd, J=5.1, 4.0, 2.2 Hz, 4H), 7.41-7.30(m, 6H), 6.79 (d, J=4.7 Hz, 1H), 5.73 (d, J=7.6 Hz, 1H), 4.94 (t, J=7.0Hz, 1H), 4.12 (td, J=4.6, 3.9 Hz, 1H), 4.05 (dd, J=4.8, 4.0 Hz, 1H),3.96 (t, J=4.7 Hz, 1H), 3.68 (ddd, J=11.8, 7.0, 4.6 Hz, 1H), 3.57-3.46(m, 1H), 3.39 (s, 3H), 1.05 (s, 8H). MS m/z: C26H33N2O6Si, [M+H]+,calculated: 497.21, measured: 497.45.

(1-2-2) Synthesis of VP-U-4:

VP-U-2 (19.84 g, 40.0 mmol), dicyclohexylcarbodiimide (DCC, 16.48 g,80.0 mmol), pyridine (4.20 g, 53.2 mmol), and trifluoroacetic acid (6.61g, 53.2 mmol) were mixed and dissolved in 200 ml of dimethyl sulfoxide(DMSO) to react under stirring at room temperature for 20 hours.Separately, tetraethyl methylenediphosphate (21.44 g, 74.4 mmol) wasdissolved in 120 ml of THF, cooled in an ice bath, added with t-BuOK(11.36 g, 101.2 mmol) at a temperature of the ice bath to react for 10min, warmed to room temperature to react for 0.5 h and added into theabove reaction solution over about 1 h. The reaction was carried out ata temperature of the ice bath for 1 h and then warmed to roomtemperature to react for 18 h. The reaction was quenched by addition ofwater. The aqueous phase isolated was extracted three times, each with200 ml of dichloromethane. All organic phases were combined and washedonce with 200 ml of saturated brine. The solvent was evaporated todryness, and the residue was purified by using a normal phase silica gelcolumn (200-300 mesh). The column was packed with petroleum ether andeluted with a gradient elution of petroleum ether:ethyl acetate=1:1-1:4.The eluate was collected, the solvent was removed by evaporation underreduced pressure, and the residue was foam-dried in a vacuum oil pump togive a total of 14.00 g of pure product VP-U-4. 1H NMR (400 MHz,DMSO-d6) δ 7.96 (d, J=7.8 Hz, 1H), 7.64 (dtd, J=5.1, 4.0, 2.2 Hz, 4H),7.41-7.30 (m, 6H), 6.82-6.71 (m, 2H), 5.90 (ddd, J=25.9, 15.0, 1.0 Hz,1H), 5.73 (d, J=7.6 Hz, 1H), 4.36-4.21 (m, 3H), 4.18 (t, J=4.9 Hz, 1H),4.05 (ddq, J=9.7, 8.5, 6.9 Hz, 2H), 3.87 (t, J=4.8 Hz, 1H), 3.39 (s,3H), 1.32 (td, J=6.9, 0.7 Hz, 6H), 1.05 (s, 8H). MS m/z: C31H42N2O8PSi,[M+H]+, calculated: 629.24, measured: 629.51.

(1-2-3) Synthesis of VP-U-5:

VP-U-4 (14.00 g, 22.29 mmol) was dissolved in 100 ml of tetrahydrofuran,added with triethylamine trihydrofluoride (17.96 g, 111.45 mmol), andstirred at room temperature for 20 hours to react completely. Thesolvent was directly evaporated to dryness and the residue was dissolvedin dichloromethane; the above evaporation and dissolution steps wereadditionally repeated twice, each with 50 ml of dichloromethane, to givea crude product. The crude product was purified by using a normal phasesilica gel column (200-300 mesh). The column was packed with petroleumether and eluted with a gradient elution of petroleum ether:ethylacetate:dichloromethane:methanol=1:1:1:0.05-1:1:1:0.25. The eluate wascollected, the solvent was removed by evaporation under reducedpressure, and the residue was foam-dried in a vacuum oil pump to give atotal of 6.70 g of pure product VP-U-5. 1H NMR (400 MHz, DMSO-d6) δ 7.96(d, J=7.8 Hz, 1H), 6.77 (dd, J=15.0, 6.2 Hz, 1H), 5.99-5.82 (m, 2H),5.73 (d, J=7.6 Hz, 1H), 5.27 (d, J=5.1 Hz, 1H), 5.10 (dd, J=5.3, 4.7 Hz,1H), 4.29 (ddq, J=9.8, 8.6, 7.0 Hz, 2H), 4.17 (ddd, J=6.2, 5.2, 1.0 Hz,1H), 4.12-3.98 (m, 3H), 3.39 (s, 2H), 1.32 (td, J=6.9, 0.6 Hz, 6H). MSm/z: C15H24N2O8P, [M+H]+, calculated: 391.13, measured: 391.38.

(1-2-4) Synthesis of VP-U-6:

VP-U-5 (391 mg, 1.0 mmol), pyridine trifluoroacetate (0.232 g, 1.2mmol), N-methylimidazole (0.099 g, 1.2 mmol), andbis(diisopropylamino)(2-cyanoethoxy)phosphine (0.452 g, 1.5 mmol) wereadded into 10 ml of anhydrous dichloromethane under argon atmosphere toreact under stirring at room temperature for 5 hours. The solvent wasevaporated to dryness, and then the residue was purified by columnchromatography (200-300 mesh normal phase silica gel, with a gradientelution of dichloromethane:acetonitrile (containing 0.5 wt %triethylamine)=3:1-1:3). The eluate was collected and concentrated toremove the solvent to give a total of 508 mg of target product VP-U-6.31P NMR (161 MHz, DMSO-d6) δ 150.34, 150.29, 17.07, 15.50. MS m/z:C24H41N4O9P2, [M+H]+, calculated: 591.23, measured: 591.55. The abovedata indicated that VP-U-6 was the target product VP-Um, which wasinvolved in the synthesis of RNA strands as a nucleoside monomer.

A 5′-phosphate ester modification was linked to 5′ terminal using thefollowing method:

As a starting material, a phosphorylated structural monomer with thefollowing structure of Formula CPR-I (provided by Suzhou GenePharmaInc., Cat #13-2601-XX) was used:

After all nucleosides of the antisense strand were linked, the monomerof Formula (CPR-I) was linked to the 5′ terminal of the antisense strandby a four-step reaction of deprotection, coupling, capping, andoxidation according to the phosphoramidite solid phase synthesis methodof nucleic acid. Then, cleavage and deprotection were performed underthe following conditions, to give the antisense strand:

The synthesized nucleotide sequence linked to the support was added into25 wt % aqueous ammonia to react at 55° C. for 16 hours, and the aqueousammonia is in an amount of 0.5 ml/μmol. The liquid was removed, and theresidue was concentrated in vacuum to dryness. After treatment withaqueous ammonia, the product was dissolved in 0.4 ml/μmolN-methylpyrrolidone, and then added with 0.3 ml/μmol of triethylamineand 0.6 ml/μmol of triethylamine trihydrofluoride, with respect to theamount of the single strand nucleic acid, for removing the 2′-TBDMSprotection on ribose. Purification and desalination: purification of thenucleic acid was achieved by using a preparative ion chromatographycolumn (Source 15Q) with a gradient elution of NaCl. Specifically,eluent A: 20 mM sodium phosphate (pH 8.1), solvent:water/acetonitrile=9:1 (v/v); eluent B: 1.5 M sodium chloride, 20 mMsodium phosphate (pH 8.1), solvent: water/acetonitrile=9:1 (v/v);elution gradient: eluent A:eluent B=100:0-50:50. The eluate wascollected, combined and desalted by using a reverse phase chromatographycolumn. The specific conditions included using a Sephadex column(filler: Sephadex-G25) for desalination and deionized water for eluting.

In the case where the target product has a 5′-phosphorothioatemodification, the same procedure as above was adopted, except that theabove oxidation reaction condition was replaced with a sulfurizationreaction condition during the linking, for performing the sulfurizationreaction.

The antisense strand of siRNA was similarly analyzed and detected usingthe same apparatus and methods as the sense strand. It was finallyconfirmed that the corresponding antisense strand of siRNA was obtained.

(1-3) Synthesis of siRNA

The S strand and the AS strand were mixed at an equimolar ratio,dissolved in water for injection and heated to 95° C., and then cooledat room temperature, such that the strands could form a duplex structurethrough hydrogen bonds.

For the sense strand and antisense strand as synthesized above, thepurity was determined by ion exchange chromatography (IEX-HPLC), and themolecular weight was analyzed by LC-MS. It was confirmed that thesynthesized nucleic acid sequences correspond to the respective siRNAsshown in Table 2.

Preparation Example 2 Preparation of Conjugate 1

In this preparation Example, siRNA conjugate (which is referred to asConjugate A1 in Table 4A) was synthesized according to the followingmethod.

(2-1) Preparation of Compound L-10:

Compound L-10 was synthesized according to the following method.

(2-1-1) Synthesis of GAL-5 (a Terminal Segment of the ConjugatingMolecule)

(2-1-1a) Synthesis of GAL-2

100.0 g of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS No.:1772-03-8, purchased from Ningbo Hongxiang Bio-Chem Co., Ltd., 463.8mmol) was dissolved in 1000 ml of anhydrous pyridine, to which 540 ml ofacetic anhydride (purchased from Enox Inc., 5565.6 mmol) was added in anice water bath to react under stirring at room temperature for 1.5hours. The resultant reaction solution was poured into 10 L of ice waterand subjected to suction filtration under reduced pressure. The residuewas washed with 2 L of ice water, and then added with a mixed solvent ofacetonitrile/toluene (v/v ratio=1:1) until completely dissolved. Thesolvent was removed by evaporation to give 130.0 g of product GAL-2 as awhite solid.

(2-1-1b) Synthesis of GAL-3

GAL-2 (35.1 g, 90.0 mmol) obtained in step (2-1-1a) was dissolved in 213ml of anhydrous 1,2-dichloroethane, to which 24.0 g of TMSOTf (CAS No.:27607-77-8, purchased from Macklin Inc., 108.0 mmol) was added in an icewater bath and nitrogen atmosphere to react at room temperatureovernight.

The reaction solution was added with 400 ml dichloromethane fordilution, filtered with diatomite, and then added with 1 L saturatedaqueous sodium bicarbonate solution and washed. An organic phase wasisolated. The aqueous phase remained was extracted twice, each with 300ml of dichloroethane, and all organic phases were combined and washedwith 300 ml of saturated aqueous sodium bicarbonate solution and 300 mlof saturated brine, respectively. The organic phase resulted fromwashing was isolated and dried with anhydrous sodium sulfate. Thesolvent was removed by evaporation under reduced pressure to give 26.9 gof product GAL-3 as a light yellow viscous syrup.

(2-1-1c) Synthesis of GAL-4

GAL-3 (26.9 g, 81.7 mmol) obtained in step (2-1-1b) was dissolved in 136ml of anhydrous 1,2-dichloroethane, added with 30 g of dry 4 Å molecularsieve powder followed by 9.0 g of 5-hexen-1-ol (CAS No.: 821-41-0,purchased from Adamas-beta Inc., 89.9 mmol), and stirred at roomtemperature for 30 minutes. 9.08 ml of TMSOTf (40.9 mmol) was added inan ice bath and nitrogen atmosphere to react under stirring at roomtemperature overnight. The 4 Å molecular sieve powder was removed byfiltration. The filtrate was added with 300 ml dichloroethane fordilution, filtered with diatomite, and then added with 500 ml ofsaturated aqueous sodium bicarbonate solution and stirred for 10 minutesfor washing. An organic phase was isolated. The aqueous phase wasextracted once with 300 ml of dichloroethane. All organic phases werecombined and washed with 300 ml of saturated aqueous sodium bicarbonatesolution and 300 ml of saturated brine respectively. The organic phaseresulted from the washing was isolated and dried with anhydrous sodiumsulfate. The solvent was removed by evaporation under reduced pressureto give 41.3 g of product GAL-4 as a yellow syrup, which was directlyused in the next oxidation reaction without purification.

(2-1-1d) Synthesis of GAL-5

GAL-4 (14.9 g, 34.7 mmol) obtained according to the method described instep (2-1-1c) was dissolved in a mixed solvent of 77 ml ofdichloromethane and 77 ml of acetonitrile, added with 103 ml ofdeionized water and 29.7 g of sodium periodate (CAS No.: 7790-28-5,purchased from Aladdin Inc., 138.8 mmol) respectively, and stirred in anice bath for 10 minutes. Ruthenium trichloride (CAS No.: 14898-67-0,available from Energy Chemical, 238 mg, 1.145 mmol) was added to reactat room temperature overnight, wherein the system temperature wascontrolled as being no more than 30° C. The resultant reaction solutionwas diluted by adding 300 ml of water under stirring, and adjusted to apH of about 7.5 by adding saturated sodium bicarbonate. The organicphase was isolated and discarded. The aqueous phase was extracted threetimes, each with 200 ml of dichloromethane, and the organic phaseresulted from the extraction was discarded. The aqueous phase resultedfrom the extraction was adjusted to a pH of about 3 with citric acidsolids and extracted three times, each with 200 ml of dichloromethane,and the resultant organic phases were combined and dried with anhydroussodium sulfate. The solvent is removed by evaporation under reducedpressure to give 6.5 g of product GAL-5 as a white foamy solid. ¹H NMR(400 MHz, DMSO) δ 12.01 (br, 1H), 7.83 (d, J=9.2 Hz, 1H), 5.21 (d, J=3.2Hz, 1H), 4.96 (dd, J=11.2, 3.2 Hz, 1H), 4.49 (d, J=8.4 Hz, 1H),4.07-3.95 (m, 3H), 3.92-3.85 (m, 1H), 3.74-3.67 (m, 1H), 3.48-3.39 (m,1H), 2.20 (t, J=6.8 Hz, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.90 (s, 3H),1.77 (s, 3H), 1.55-1.45 (m, 4H).

(2-1-2) Synthesis of M-11-T3:

J-0 (1.883 g, 10 mmol, purchased from Alfa Aesar) was dissolved in 25 mlof acetonitrile, added with triethylamine (4.048 g, 40 mmol), and cooledto 0° C. in an ice water bath. Ethyl trifluoroacetate (5.683 g, 40 mmol)was added to react at room temperature for 22 hours. The solvent wasremoved by evaporation under reduced pressure, and the residue wasfoam-dried in a vacuum oil pump for 18 hours to give 5.342 g of crudesolid product M-11-T3, which was directly used in subsequent reactionwithout further purification. MS m/z: C15H22F9N4O3, [M+H]+, calculated:477.35, measured: 477.65.

(2-1-3) Synthesis of M-11-T3-Tr:

The crude product M-11-T3 (5.342 g, 10 mmol) was dissolved in 50 ml ofdichloromethane. The resultant reaction solution was added with TrCl(3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) to react understirring at room temperature for 20 hours. The reaction solution waswashed twice, each with 20 ml of saturated sodium bicarbonate and oncewith 20 ml of saturated brine. The resultant organic phase was driedwith anhydrous sodium sulfate and filtered. The organic solvent wasremoved by evaporation under reduced pressure, and the residue wasfoam-dried in a vacuum oil pump overnight to give 7.763 g of crude solidproduct M-11-T3-Tr. MS m/z: C34H36F9N4O3, [M+Na]+, calculated: 741.25,measured: 741.53. The crude solid product M-11-T3-Tr was then used inthe next step for synthesis of M-18-Tr without purification.

(2-1-4) Synthesis of M-18-Tr:

The crude product M-11-T3-Tr (7.763 g, 10 mmol) obtained in step (2-1-3)was dissolved in 100 ml of methanol, and added with 100 ml of aqueousmethylamine solution (40 mass %) to react under stirring at 50° C. for23 hours. Insoluble particles were removed by filtration. The solventwas removed by evaporation under reduced pressure, and the residue wasadded with 200 ml of mixed solvent of DCM:methanol in a volume ratio of1:1, washed with 50 ml of saturated sodium bicarbonate. The aqueousphase obtained was extracted three times, each with 50 ml ofdichloromethane. All organic phases were combined, dried with anhydroussodium sulfate and filtered. The solvent was removed by evaporationunder reduced pressure, and the residue was foam-dried in a vacuum oilpump overnight, and purified by using a normal phase silica gel column(200-300 mesh). The column was packed with petroleum ether and addedwith 1 wt % triethylamine for neutralizing the acidity of silica gel,and eluted with a gradient elution of dichloromethane:methanol:aqueousammonia (25 wt %)=1:1:0.05-1:1:0.25. The eluate was collected, thesolvent was removed by evaporation under reduced pressure, and theresidue was foam-dried in a vacuum oil pump to give 2.887 g of pureproduct M-18-Tr. ¹H NMR (400 MHz, DMSO) δ7.47-7.39 (m, 6H), 7.32-7.24(m, 6H), 7.19-7.12 (m, 3H), 2.60-2.47 (m, 4H), 2.46-2.19 (m, 13H),1.70-1.55 (m, 4H), 1.40 (p, J=6.8 Hz, 2H). MS m/z: C₂₈H₃₉N₄, [M+H]+,calculated: 431.65, measured: 432.61.

(2-1-5) Synthesis of L-5-Tr:

M-18-Tr (2.02 g, 4.69 mmol) obtained in step (2-1-4) and GAL-5 (6.93 g,15.48 mmol) obtained in step (2-1-1) were mixed and dissolved in 47 mlof acetonitrile, and added with N-methylmorpholine (3.13 g, 30.96 mmol)and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride(DMTMM, 4.28 g, 15.48 mmol) to react under stirring at room temperaturefor 2 hours. The resultant reaction solution was diluted with 200 ml ofdichloromethane. The organic phase was washed with 100 ml of a saturatedsodium bicarbonate solution and 100 ml of saturated brine, dried withanhydrous sodium sulfate, and filtered. Then the solvent was removed byevaporation under reduced pressure to give a crude product. The crudeproduct was purified by using a normal phase silica gel column (200-300mesh). The column was packed with petroleum ether, added with 1 wt %triethylamine for neutralizing the acidity of silica gel, and elutedwith a gradient elution of dichloromethane:methanol=100:5-100:7. Theeluate was collected, and evaporated to dryness under reduced pressureto give 7.49 g of pure product L-5-Tr. ¹H NMR (400 MHz, DMSO) δ7.83-7.10(m, 4H), 7.67-7.60 (m, 1H), 7.44-7.34 (m, 6H), 7.33-7.24 (m, 6H),7.20-7.15 (m, 3H), 5.22 (s, 3H), 4.97 (d, J=11.3 Hz, 3H), 4.49 (d, J=8.4Hz, 3H), 4.06-3.07 (m, 9H), 3.95-3.83 (m, 3H), 3.77-3.64 (m, 3H),3.45-3.35 (m, 3H), 3.12-2.87 (m, 8H), 2.30-2.15 (m, 3H), 2.11-1.98 (m,22H), 1.95-1.84 (m, 11H), 1.81-1.61 (m, 14H), 1.54-1.36 (m, 14H). MSm/z: C₈₅H119N7030, [M+H]+, calculated: 1718.81, measured: 1718.03.

(2-1-6) Synthesis of L-8:

L-5-Tr (5.94 g, 3.456 mmol) obtained in step (2-1-5) was dissolved in 69ml of dichloromethane, and added with dichloroacetic acid (13.367 g,103.67 mmol) to react at room temperature for 2 hours. The resultantreaction solution was diluted by adding 100 ml of dichloromethane,washed and adjusted to pH 7-8 with saturated sodium bicarbonatesolution. The aqueous phase isolated was extracted six times, each with30 ml of dichloromethane. All organic phases were combined, dried withanhydrous sodium sulfate, and filtered. Then the solvent was removed byevaporation under reduced pressure to give a crude product. The crudeproduct was purified by using a normal phase silica gel column (200-300mesh). The column was added with 10 wt % triethylamine for neutralizingthe acidity of silica gel and equilibrated with 1 wt ‰ triethylamine,and eluted with a gradient elution ofdichloromethane:methanol=100:30-100:40. The eluate was collected, andthe solvent was removed by evaporation under reduced pressure to give4.26 g of pure product L-8. ¹H NMR (400 MHz, DMSO) δ 7.84 (d, J=9.0 Hz,3H), 7.27-7.23 (m, 1H), 7.13-7.18 (m, 1H), 5.22 (d, J=3.1 Hz, 3H), 4.97(dd, J=11.3, 3.1 Hz, 3H), 4.48 (d, J=8.4 Hz, 3H), 4.09-3.98 (m, 9H),3.88 (dd, J=19.3, 9.3 Hz, 3H), 3.75-3.66 (m, 3H), 3.44-3.38 (m, 3H),3.17-3.30 (m, 4H), 3.10-2.97 (m, 4H), 2.35-2.20 (m, 6H), 2.15-2.08 (m,9H), 2.07-1.98 (m, 13H), 1.94-1.87 (m, 9H), 1.81-1.74 (m, 9H), 1.65-1.42(m, 18H). MS m/z: C85H119N7O30, [M+H]+, calculated: 1477.59, measured:1477.23.

(2-1-7a) Synthesis of A-1

DMTrCl (4,4′-dimethoxytrityl chloride, 38.12 g, 112.5 mmol) wasdissolved in 450 ml of anhydrous pyridine, and added with calciumDL-glycerate hydrate (12.88 g, 45.0 mmol) to react at 45° C. for 22hours. The resultant reaction solution was filtered. The residue wasrinsed with 200 ml of DCM, and the filtrate was concentrated to drynessunder reduced pressure. The residue was redissolved in 500 ml ofdichloromethane and washed twice, each with 200 ml of 0.5 Mtriethylamine phosphate (pH=7-8). The aqueous phase isolated wasextracted twice, each with 200 ml of dichloromethane. All organic phaseswere combined, dried with anhydrous sodium sulfate, and filtered. Thesolvent was removed by evaporation under reduced pressure, and theresidue was purified by using a normal phase silica gel column (200-300mesh) which was eluted with a gradient elution of petroleum ether:ethylacetate:dichloromethane:methanol=1:1:1:0.35-1:1:1:0.55. The eluate wascollected, and the solvent was removed by evaporation under reducedpressure. The residue was redissolved in 500 ml of dichloromethane, andwashed once with 200 ml of 0.5 M triethylamine phosphate. The aqueousphase isolated was extracted twice, each with 200 ml of dichloromethane.All organic phases were combined, dried with anhydrous sodium sulfate,and filtered. The solvent was removed by evaporation under reducedpressure (reduced pressure in a vacuum oil pump) to dryness overnight togive 20.7 g of product A-1 as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ7.46 (ddd, J=6.5, 2.3, 1.1 Hz, 1H), 7.40-7.28 (m, 7H), 6.89-6.81 (m,4H), 4.84 (d, J=5.0 Hz, 1H), 4.36-4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd,J=12.4, 7.0 Hz, 1H), 3.67 (dd, J=12.3, 7.0 Hz, 1H), 2.52 (q, J=6.3 Hz,6H), 1.03 (t, J=6.3 Hz, 9H). MS m/z: C24H23O6, [M−H]−, calculated:407.15, measured: 406.92.

(2-1-7b) Synthesis of L-7:

L-8 (2.262 g, 1.532 mmol) obtained in step (2-1-6) and A-1 (2.342 g,4.596 mmol) obtained in step (2-1-7a) were mixed and dissolved in 16 mlof dichloromethane, added with3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one (DEPBT, 1.375 g,4.596 mmol), and further added with diisopropylethylamine (1.188 g,9.191 mmol) to react under stirring at 25° C. for 2 hours. The organicphase was washed with 10 ml of saturated sodium bicarbonate. The aqueousphase isolated was extracted three times, each with 10 ml ofdichloromethane. All organic phases were combined and washed with 10 mlof saturated brine, and the aqueous phase isolated was extracted twice,each with 10 ml of dichloromethane, and the obtained organic phases werecombined, dried with anhydrous sodium sulfate and filtered. The solventwas removed by evaporation under reduced pressure, and the residue wasfoam-dried in a vacuum oil pump overnight to give 4.900 g of crudeproduct. The crude product was subjected to a column purification. Thecolumn was filled with 120 g normal phase silica gel (200-300 mesh),added with 20 ml triethylamine for neutralizing the acidity of silicagel, equilibrated with petroleum ether containing 1 wt % triethylamine,and eluted with a gradient elution of petroleum ether:ethylacetate:dichloromethane:N,N-dimethylformamide=1:1:1:0.5-1:1:1:0.6. Theeluate was collected, and the solvent was removed by evaporation underreduced pressure to give 2.336 g of pure product L-7. ¹H NMR (400 MHz,DMSO) δ7.90-7.78 (m, 4H), 7.75-7.64 (m, 1H), 7.38-7.18 (m, 9H),6.91-6.83 (m, 4H), 5.25-5.10 (m, 4H), 4.97 (dd, J=11.2, 3.2 Hz, 3H),4.48-4.30 (m, 4H), 4.02 (s, 9H), 3.93-3.84 (m, 3H), 3.76-3.66 (m, 9H),3.45-3.35 (m, 3H), 3.24-2.98 (m, 10H), 2.30-2.20 (m, 2H), 2.11-1.88 (m,31H), 1.80-1.40 (m, 28H). MS m/z: C90H128N7O35, [M−DMTr]+, calculated:1564.65, measured: 1564.88.

(2-1-8) Synthesis of L-9:

L-7 (2.300 g, 1.26 mmol) obtained in step (2-1-7b), succinic anhydride(0.378 g, 3.78 mmol) and 4-dimethylaminopyridine (DMAP, 0.462 g, 3.78mmol) were mixed and dissolved in 13 ml of dichloromethane, furtheradded with DIPEA (0.814 g, 6.30 mmol), and stirred at 25° C. for 24hours. The resultant reaction solution was washed with 5 ml of 0.5 Mtriethylamine phosphate. The aqueous phase isolated was extracted threetimes, each with 5 ml of dichloromethane. All organic phases werecombined and removed by evaporation under reduced pressure to give 2.774g of crude product. The crude product was subjected to a columnpurification. The column was filled with 60 g normal phase silica gel(200-300 mesh), added with 1 wt % triethylamine for neutralizing theacidity of silica gel, equilibrated with dichloromethane and eluted witha gradient elution of 1 wt ‰ triethylamine-containingdichloromethane:methanol=100:18-100:20. The eluate was collected, andthe solvent was removed by evaporation under reduced pressure to give1.874 g of pure product of L-9 conjugating molecule (compoundconjugating molecule 1). ¹H NMR (400 MHz, DMSO) δ 8.58 (d, J=4.2 Hz,1H), 7.94-7.82 (m, 3H), 7.41-7.29 (m, 5H), 7.22 (d, J=8.1 Hz, 5H), 6.89(d, J=8.3 Hz, 4H), 5.49-5.37 (m, 1H), 5.21 (d, J=3.0 Hz, 3H), 4.97 (d,J=11.1 Hz, 3H), 4.49 (d, J=8.2 Hz, 3H), 4.02 (s, 9H), 3.88 (dd, J=19.4,9.4 Hz, 3H), 3.77-3.65 (m, 9H), 3.50-3.39 (m, 6H), 3.11-2.90 (m, 5H),2.61-2.54 (m, 4H), 2.47-2.41 (m, 2H), 2.26-2.17 (m, 2H), 2.15-1.95 (m,22H), 1.92-1.84 (m, 9H), 1.80-1.70 (m, 10H), 1.65-1.35 (m, 17H),1.31-1.19 (m, 4H), 0.96 (t, J=7.1 Hz, 9H). MS m/z: C94H132N7O38,[M−DMTr]+, calculated: 1664.72, measured: 1665.03.

(2-1-9) Synthesis of compound L-10:

In this step, a compound L-10 was prepared by linking the L-9conjugating molecule to a solid phase support.

The L-9 conjugating molecule (0.233 g, 0.1126 mmol) obtained in step(1-1-8), O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU,0.064 g, 0.1689 mmol) and diisopropylethylamine (DIPEA, 0.029 g, 0.2252mmol) were mixed and dissolved in 19 ml of acetonitrile, and stirred atroom temperature for 5 minutes. The resultant reaction solution wasadded with Aminomethyl resin (H₂NResin, 0.901 g, 100-200 mesh, aminoloading: 400 μmol/g, purchased from Tianjin Nankai HECHENG S&T Co.,Ltd.). A reaction was performed on a shaker at 25° C. and 220 rpm/minfor 15 hours, and the reaction mixture was filtered. The residue wasrinsed twice (each with 30 ml of DCM), three times (each with 30 ml ofacetonitrile), and once (with 30 ml of ethyl ether), and dried in avacuum oil pump for 2 hours. Then starting materials (CapA, CapB,4-dimethylaminopyridine (DMAP) and acetonitrile) were added according tothe charge ratios shown in Table 3 for a capping reaction. The reactionwas performed on a shaker at 25° C. and 200 rpm/min for 5 hours. Thereaction solution was filtered. The residue was rinsed three times, eachwith 30 ml of acetonitrile, subjected to suction filtration to dryness,and dried overnight under a reduced pressure in a vacuum oil pump togive 1.100 g of compound L-10 (i.e., L-9 conjugating molecule linked toa solid phase support), with a loading of 90.8 μmol/g.

TABLE 3 The charge ratios of capping reaction Starting Materials AmountSpecs Lot No. Manufacturer CapA 20 ml — — — CapB 2.3 ml — — — DMAP 0.01g analytical pure I1422139 Aladdin acetonitrile 2.3 ml spectroscopicO15161001 CINC pure (Shanghai) Co., Ltd

In the above table, CapA and CapB are solutions of capping agents. CapAis a solution of 20% by volume of N-methylimidazole in a mixture ofpyridine/acetonitrile, wherein the volume ratio of pyridine toacetonitrile is 3:5. CapB is a solution of 20% by volume of aceticanhydride in acetonitrile.

In the following synthesis, the sequences of the sense strand andantisense strand correspond to S Sequence and AS Sequence of Conjugate 1as shown in Table 4, respectively.

(2-2) Synthesis of a Sense Strand

Nucleoside monomers were linked one by one in 3′ to 5′ directionaccording to the arrangement sequence of nucleotides in the sense strandby the phosphoramidite solid phase synthesis method, starting the cyclesfrom the Compound L-10 prepared in the above step. The linking of eachnucleoside monomer included a four-step reaction of deprotection,coupling, capping, and oxidation or sulfurization. Therein, when twonucleotides is linked via a phosphoester linkage, a four-step reactionof deprotection, coupling, capping, and oxidation was included duringlinking of the later nucleoside monomer; and when two nucleotides islinked via a phosphorothioate linkage, a four-step reaction ofdeprotection, coupling, capping, and sulfurization was included duringlinking of the later nucleoside monomer. The conditions of the abovereactions were the same as those used for the synthesis of the sensestrand in Preparation Example 1 as described above.

(2-3) Synthesis of an Antisense Strand

An antisense strand AS of Conjugate 1 was synthesized by starting thecycles using a universal solid phase support (UnyLinker™ loadedNittoPhaseRHL Solid Supports, Kinovate Life Sciences Inc.) via thephosphoramidite solid phase synthesis method. The reaction conditions ofdeprotection, coupling, capping, oxidation or sulfurization, cleavage,deprotection, purification and desalination in the solid phase synthesismethod were the same as those used for the synthesis of the antisensestrand in Preparation Example 1 as described above.

After completion of the synthesis, for the sense strand and antisensestrand synthesized above, the purity was determined by ion exchangechromatography (IEX-HPLC), and the molecular weight was analyzed byLC-MS. The measured molecular weight values were compared with thecalculated values for validating the synthesized sense strand andantisense strand.

(2-4) Synthesis of Conjugate A1

The S strand and AS strand were respectively dissolved in water forinjection to give a solution of 40 mg/m. They are mixed at an equimolarratio, heated at 50° C. for 15 min, and then cooled at room temperature,such that they could form a double stranded structure via hydrogenbonds. The conjugate was diluted to a concentration of 0.2 mg/mL withultra-pure water (prepared by Milli-Q ultra-pure water instrument, withresistivity of 18.2MΩ*cm (25° C.)). The molecular weight was measured byLC-MS instrument (purchased from Waters Corp., model: LCT Premier). As aresult, the calculated values of the molecular weight for S and AS were7516.37 and 7061.57 respectively, and the measured values thereof were7516.6 and 7060.49 respectively. Since the measured values were inconformity with the calculated values, it was indicated that the targetConjugate A1 with a structure as shown by Formula (403) was obtained.

Preparation Example 3 Preparation of the Conjugates of the PresentDisclosure and Comparative Conjugates

Conjugates A2-A7, B1-B2, C₂, C₁₂-C₁₃, D2, D12-D13, E1-E4, F1-F3, G1-G3and Comparative Conjugates A2, C₁, D1, E1, F1, G1 listed in Tables 4A-4Gwere synthesized by using the same methods as those in PreparationExample 2, and it is expected that Conjugates A8-A11, B3-B7, C1, C3, D1,D3, E5-E9, F4-F11, G4-G9 listed in Tables 4A-4G can be prepared, exceptthat: the siRNA sequences of these conjugates were the correspondingsequences shown in Tables 4A-4G, respectively. After completion of thesynthesis, the resultant conjugates were confirmed by using the samedetection methods as those in Preparation Example 2.

Conjugate A2: Calculated values S:7516.37, AS:7065.58, Measured valuesS:7516.6, AS:7064.5;

Conjugate A3: Calculated values S:7504.34, AS:7139.68, Measured valuesS:7515.6, AS:7138.9;

Conjugate A4: Calculated values S:7516.37, AS:7081.64, Measured valuesS:7515.6, AS:7080.9;

Conjugate A5: Calculated values S:8218.83, AS:7703.05, Measured valuesS:8218, AS:7702.5;

Conjugate A6: Calculated values S:7516.37, AS:6985.58, Measured valuesS:7516.5, AS:6984.9;

Conjugate B1: Calculated values S:7407.22, AS:7208.77, Measured valuesS:7406.4, AS:7208.1;

Conjugate B2: Calculated values S:7407.22, AS:7170.72, Measured valuesS:7406.5, AS:7170.1,

Conjugate C2: Calculated values S:7485.3, AS:7161.7, Measured valuesS:7484.4, AS:7160.9;

Conjugate D2: Calculated values S:7423.22, AS:7207.78, Measured valuesS:7422.6, AS:7207.2;

Conjugate F2: Calculated values S:7649.55, AS:6995.47, Measured valuesS:7648.8, AS:6994.8;

Conjugate F3: Calculated values S:7649.55, AS:7011.53, Measured valuesS:7648.8, AS:7010.9;

Conjugate E1: Calculated values S:7584.5, AS:7007.46, Measured valuesS:7584, AS:7006.2;

Conjugate E2: Calculated values S:7584.5, AS:7011.47, Measured valuesS:7584, AS:7011.3;

Conjugate E4: Calculated values S:7572.47, AS:6907.41, Measured valuesS:7571.8, AS:6906.9;

Since the measured values of the molecular weight are in conformity withthe calculated values, it is indicated that the target conjugates areobtained. These conjugates all have a structure as shown by Formula(403).

Table 4 siRNA conjugates

TABLE 4A SEQ Example No Sequence Direction: 5′-3′ ID NO ConjugateL10-siHBa1M1SVP S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 153 A1 UmUmCmAmAmAm ASVP-UmsUfsUmGmAmAfGmUmAmUmGmC 154 mCmUfCmAfAmGmGmsUmsUm ConjugateL10-siHBa1M1SP S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 155 A2 UmUmCmAmAmAm ASP-UmsUfsUmGmAmAfGmUmAmUmGmCm 156 CmUfCmAfAmGmGmsUmsUm ConjugateL10-siHBa1M1SPsT S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 157 A3 UmUmCmAmAmAm ASPs-TmsUfsUmGmAmAfGmUmAmUmGmCm 158 CmUfCmAfAmGmGmsUmsUm ConjugateL10-siHBa1M1SPs S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 159 A4 UmUmCmAmAmAm ASPs-UmsUfsUmGmAmAfGmUmAmUmGmCm 160 CmUfCmAfAmGmGmsUmsUm ConjugateL10-siHBa2M1S S GmsAmsCmCmUmUmGmAmGfGfCfAmUm 161 A5 AmCmUmUmCmAmAmAm ASUmsUfsUmGmAmAfGmUmAmUmGmCmC 162 mUfCmAfAmGmGmUmCmsGmsGm ConjugateL10-siHBa1M1S S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 163 A6 UmUmCmAmAmAm ASUmsUfsUmGmAmAfGmUmAmUmGmCmC 164 mUfCmAfAmGmGmsUmsUm ConjugateL10-siHBa2M1SVP S GmsAmsCmCmUmUmGmAmGfGfCfAmUm 165 A7 AmCmUmUmCmAmAmAmAS VP-UmsUfsUmGmAmAfGmUmAmUmGmC 166 mCmUfCmAfAmGmGmUmCmsGmsGm ConjugateL10-siHBa1M1 S CmCmUmUmGmAmGfGfCfAmUmAmCmU 167 A8 mUmCmAmAmAm ASUmUfUmGmAmAfGmUmAmUmGmCmCm 168 UfCmAfAmGmGmUmUm Conjugate L10-siHBa2M1 SGmAmCmCmUmUmGmAmGfGfCfAmUmA 169 A9 mCmUmUmCmAmAmAm ASUmUfUmGmAmAfGmUmAmUmGmCmCm 170 UfCmAfAmGmGmUmCmGmGm ConjugateL10-siHBa1M1VP S CmCmUmUmGmAmGfGfCfAmUmAmCmU 171 A10 mUmCmAmAmAm ASVP-UmUfUmGmAmAfGmUmAmUmGmCm 172 CmUfCmAfAmGmGmUmUm ConjugateL10-siHBa2M1VP S GmAmCmCmUmUmGmAmGfGfCfAmUmA 173 A11 mCmUmUmCmAmAmAm ASVP-UmUfUmGmAmAfGmUmAmUmGmCm 174 CmUfCmAfAmGmGmUmCmGmGm ConjugateP10-siHBa1M1SVP S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 175 A12 UmUmCmAmAmAm ASVP-UmsUfsUmGmAmAfGmUfAmUmGmCm 176 CmUfCmAfAmGmGmsUmsUm ConjugateR5-siHBa1M1SVP S CmCmUmUmGfAmGfGfCfAmUmAmCmUm 177 A13 UmCmAmAmAm ASVP-UmUfUmGmAmAfGmUfAfUmGmCmC 178 mUfCmAfAmGmGmUmUm ConjugateLA5-siHBa1M1SVP S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 179 A14 UmUmCmAmAmAm ASVP-UmsUfsUmGmAmAfGmUmAmUmGmC 180 mCmUfCmAfAmGmGmsUmsUm ConjugateLB5-siHBa1M1SVP S CmCmUmUmGfAmGfGfCfAmUmAmCmUm 181 A15 UmCmAmAmAm ASVP-UmUfUmGmAmAfGmUfAfUmGmCmC 182 mUfCmAfAmGmGmUmUm ConjugateV8-siHBa1M1SVP S CmCmUmUmGfAmGfGfCfAmUmAmCmUm 183 A16 UmCmAmAmAm ASVP-UmUfUmGmAmAfGmUfAfUmGmCmC 184 mUfCmAfAmGmGmUmUm ConjugateW8-siHBa1M1SVP S CmCmUmUmGfAmGfGfCfAmUmAmCmUm 185 A17 UmCmAmAmAm ASVP-UmUfUmGmAmAfGmUfAfUmGmCmC 186 mUfCmAfAmGmGmUmUm ConjugateX8-siHBa1M1SVP S CmCmUmUmGfAmGfGfCfAmUmAmCmUm 187 A18 UmCmAmAmAm ASVP-UmUfUmGmAmAfGmUfAfUmGmCmC 188 mUfCmAfAmGmGmUmUm ConjugateZ5-siHBa1M1SVP S CmCmUmUmGfAmGfGfCfAmUmAmCmUm 189 A19 UmCmAmAmAm ASVP-UmUfUmGmAmAfGmUfAfUmGmCmC 190 mUfCmAfAmGmGmUmUm ConjugateFIN-siHBa2M1SVP S GmsAmsCmCmUmUmGmAmGfGfCfAmUm 191 A20 AmCmUmUmCmAmAmAmAS VP-UmsUfsUmGmAmAfGmUmAmUmGmC 192 mCmUfCmAfAmGmGmUmCmsGmsGm ConjugateFIN-siHBa1M1SVP S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 193 A21 UmUmCmAmAmAm ASVP-UmsUfsUmGmAmAfGmUmAmUmGmC 194 mCmUfCmAfAmGmGmsUmsUm ConjugateFIN-siHBa2M1S S GmsAmsCmCmUmUmGmAmGfGfCfAmUm 195 A22 AmCmUmUmCmAmAmAm ASUmsUfsUmGmAmAfGmUmAmUmGmCmC 196 mUfCmAfAmGmGmUmCmsGmsGm ConjugateFIN-siHBa1M1S S CmsCmsUmUmGmAmGfGfCfAmUmAmCm 197 A23 (X2UR8 NON-VP)UmUmCmAmAmAm AS UmsUfsUmGmAmAfGmUmAmUmGmCmC 198 mUfCmAfAmGmGmsUmsUmComparative FIN-siHBa1M3 S CmCmUmUmGAGGCmAUmACmUmUmCm 199 ConjugateAAAdT-S-dT A1 AS UfUmUfGAAGUfAUGCCUfCAAGGdT-S-dT 200 ComparativeL10-siHBa1M2SVP S CmsCmsUmUmGfAmGfGfCfAmUmAmCmU 201 ConjugatemUmCmAmAmAm A2 AS VP-UmsUfsUmGmAmAfGmUfAfUmGmCm 202 CmUfCmAfAmGmGmsUmsUmComparative AD-66810 S GmsUmsGmUmGfCmAfCfUfUmCmGmCmU 203 ConjugatemUmCmAmCmAm A3 AS UmsGfsUmGmAmAfGmCfGfAmAmGmUmG 204 fCmAfCmAmCmsUmsUm

TABLE 4B SEQ ID Conjugate No Sequence Direction: 5′-3′ NO ConjugateL10-siHBb1M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 205 B1 CmUmUmCmUmAm ASUmsAfsGmAmAmGfAmUmGmAmGmGmC 206 mAfUmAfGmCmAmsGmsCm ConjugateL10-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 207 B2 CmUmUmCmUmAm ASUmsAfsGmAmAmGfAmUmGmAmGmGmC 208 mAfUmAfGmCmAmsUmsUm ConjugateL10-siHBb1M1S S UmGmCmUmAfUmGfCfCfUmCmAmUmCm 209 B3 UmUmCmUmAm ASUmAfGmAmAmGfAmUfGfAmGmGmCmAf 210 UmAfGmCmAmGmCm Conjugate L10-siHBb2M1SS UmGmCmUmAfUmGfCfCfUmCmAmUmCm 211 B4 UmUmCmUmAm ASUmAfGmAmAmGfAmUfGfAmGmGmCmAf 212 UmAfGmCmAmUmUm ConjugateL10-siHBb4M1SVP S GmsCmsUmGmCmUmAmUmGfCfCfUmCm 213 B5 AmUmCmUmUmCmUmAmAS VP-UmsAfsGmAmAmGfAmUmGmAmGmG 214 mCmAfUmAfGmCmAmGmCmsGmsCm ConjugateL10-siHBb1M1SP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 215 B6 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 216 mCmAfUmAfGmCmAmsUmsUm ConjugateL10-siHBb1M1SPs S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 217 B7 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 218 mCmAfUmAfGmCmAmsUmsUm ConjugateP10-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 219 B8 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 220 mCmAfUmAfGmCmAmsUmsUm ConjugateR5-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 221 B9 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 222 mCmAfUmAfGmCmAmsUmsUm ConjugateLA5-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 223 B10 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 224 mCmAfUmAfGmCmAmsUmsUm ConjugateLB5-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 225 B11 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 226 mCmAfUmAfGmCmAmsUmsUm ConjugateV8-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 227 B12 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 228 mCmAfUmAfGmCmAmsUmsUm ConjugateW8-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 229 B13 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 230 mCmAfUmAfGmCmAmsUmsUm ConjugateX8-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 231 B14 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 232 mCmAfUmAfGmCmAmsUmsUm ConjugateZ5-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUm 233 B15 CmUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmG 234 mCmAfUmAfGmCmAmsUmsUm ConjugateFIN-siHBb1M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUmC 235 B16 mUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmGm 236 CmAfUmAfGmCmAmsGmsCm ConjugateFIN-siHBb2M1SVP S UmsGmsCmUmAmUmGfCfCfUmCmAmUmC 237 B17 mUmUmCmUmAm ASVP-UmsAfsGmAmAmGfAmUmGmAmGmGm 238 CmAfUmAfGmCmAmsUmsUm ComparativeFIN-NC S UUCUCCGAACGUGUCACGU 239 Conjugate AS ACGUGACACGUUCGGAGAAUU 240B1

TABLE 4C SEQ NO Conjugate No Sequence direction 5′-3′ NO ConjugateL10-siHBc1M1 S UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCm 241 C1 UmGmAm ASUmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCf 242 AmGmAmCmGm ConjugateL10-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 243 C2 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 244 AmCfAmGmAmsCmsGm ConjugateL10-siHBc2M1SVP S CmsGmsUmCmUmGmUmGmCfCfUfUmCmUmCmA 245 C3 mUmCmUmGmAmAS VP-UmsCfsAmGmAmUfGmAfGfAmAmGmGmCfA 246 mCfAmGmAmsCmsGmGmGm ConjugateP10-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 247 C4 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 248 AmCfAmGmAmsCmsGm ConjugateR5-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 249 C5 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 250 AmCfAmGmAmsCmsGm ConjugateLA5-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 251 C6 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 252 AmCfAmGmAmsCmsGm ConjugateLB5-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 253 C7 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 254 AmCfAmGmAmsCmsGm ConjugateV8-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 255 C8 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 256 AmCfAmGmAmsCmsGm ConjugateW8-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 257 C9 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 258 AmCfAmGmAmsCmsGm ConjugateX8-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 259 C10 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 260 AmCfAmGmAmsCmsGm ConjugateZ5-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 261 C11 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 262 AmCfAmGmAmsCmsGm ConjugateL10-siHBc1M1SP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 263 C12 mUmGmAm ASP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCfA 264 mCfAmGmAmsCmsGm ConjugateL10-siHBc1M1SPs S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 265 C13 mUmGmAm ASPs-UmsCfsAmGmAmUfGmAmGmAmamGmGmCfA 266 mCfAmGmAmsCmsGm ConjugateFIN-siHBc1M1SVP S UmsCmsUmGmUmGmCfCfUfUmCmUmCmAmUmC 267 C14 mUmGmAm ASVP-UmsCfsAmGmAmUfGmAmGmAmAmGmGmCf 268 AmCfAmGmAmsCmsGm ComparativeL10-NC S GUGUGCACUUCGCUUCACA 269 Conjugate AS UGUGAAGCGAAGUGCACACUU 270C1

TABLE 4D Sequence SEQ Direction ID Conjugate No 5′-3′ NO Conjugate L10-S CmGmUmGmUm 271 D1 SiHBd1M1 GmCfAfCfUm UmCmGmCmUm UmCmAmAm ASUmUfGmAmAm 272 GfCmGmAmAm GmUmGmCfAm CfAmCmGmGm Um Conjugate L10- SCmsGmsUmGm 273 D2 SiHBd1M1 UmGmCfAfCf SVP UmUmCmGmCm UmUmCmAmAm ASVP-UmsUfsG 274 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUm Conjugate L10-S AmsCmsCmGm 275 D3 SiHBd2M1 UmGmUmGmCf SVP AfCfUmUmCm GmCmUmUmCm AmAmAS VP-UmsUfsG 276 mAmAmGfCm GmAmAmGmUm GmCfAmCfAm CmGmGmUmsC msCmConjugate P10- S CmsGmsUmGm 277 D4 siHBd1M1 UmGmCfAfCf SVP UmUmCmGmCmUmUmCmAmAm AS VP-UmsUfsG 278 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmConjugate R5- S CmsGmsUmGm 279 D5 s1HBd1M1 UmGmCfAfCf SVP UmUmCmGmCmUmUmCmAmAm AS VP-UmsUfsG 280 mAmAmGfCmG mAmAmGmUmG mCfAmCmAmC mGmsGmsUmConjugate LA5- S CmsGmsUmGm 281 D6 siHBd1M UmGmCfAfCf 1SVP UmUmCmGmCmUmUmCmAmAm AS VP-UmsUfsG 282 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmConjugate LB5-siHBd1M1 S CmsGmsUmGm 283 D7 SVP UmGmCfAfCf UmUmCmGmCmUmUmCmAmAm AS VP-UmsUfsG 284 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmConjugate V8- S CmsGmsUmGm 285 D8 siHBd1M1 UmGmCfAfCf SVP UmUmCmGmCmUmUmCmAmAm AS VP-UmsUfsG 286 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmConjugate W8- S CmsGmsUmGm 287 D9 siHBd1M UmGmCfAfCf 1SVP UmUmCmGmCmUmUmCmAmAm AS VP-UmsUfsG 288 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmConjugate X8- S CmsGmsUmGm 289 D10 siHBd1M1 UmGmCfAfCf SVP UmUmCmGmCmUmUmCmAmAm AS VP-UmsUfsG 290 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmConjugate Z5- S CmsGmsUmGm 291 D11 siHBd1M1 UmGmCfAfCf SVP UmUmCmGmCmUmUmCmAmAm AS VP-UmsUfsG 292 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmConjugate L10- S CmsGmsUmGm 293 D12 siHBd1M UmGmCfAfCf 1SP UmUmCmGmCmUmUmCmAmAm AS P-UmsUfsGm 294 AmAmGfCmGm AmAmGmUmGm CfAmCfAmCm GmsGmsUmConjugate L10- S CmsGmsUmGm 295 D13 siHBd1M UmGmCfAfCf 1SPs UmUmCmGmCmUmUmCmAmAm AS Ps-UmsUfsG 296 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmConjugate FIN- S CmsGmsUmGm 297 D14 siHBd1M UmGmCfAfCf 1SVP UmUmCmGmCmUmUmCmAmA m AS VP-UmsUfsG 298 mAmAmGfCmG mAmAmGmUmG mCfAmCfAmC mGmsGmsUmComparative L10-NC S GUGUGCACUU 299 Conjugate CGCUUCACA D1 AS UGUGAAGCGA300 AGUGCACACU U

TABLE 4E Sequence SEQ Direction ID Conjugate No 5′-3′ NO Conjugate L10-S CmsCmsAmAm 301 E1 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 302 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate L10-S CmsCmsAmAm 303 E2 siAN1M3SP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASP-UmsAfsGm 304 UmUmCfUmUm GmGmUmGmCm UfCmUfUmGm GmsCmsUm Conjugate L10-S CmsCmsAmAm 305 E3 siAN1M3SPs GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASPs-UmsAfsG 306 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate L10-S CmsCmsAmAm 307 E4 siAN1M3S GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASUmsAfsGmUm 308 UmCfUmUmGm GmUmGmCmUf CmUfUmGmGm sCmsUm Conjugate L10- SCmCmAmAmGm 309 E5 siAN1M3VP AmGfCfAfCm CmAmAmGmAm AmCmUmAm AS VP-UmAfGmU310 mUmCfUmUmG mGmUmGmCmU fCmUfUmGmG mCmUm Conjugate L10- S AmsGmsCmCm311 E6 siAN2M2S AmAmGfAmGf CfAfCmCmAm AmGmAmAmCm UmAm AS UmsAfsGmUm 312UmCfUmUmGm GmUmGmCmUf CmUfUmGmGm CmUmsUmsGm Conjugate L10- S AmsGmsCmCm313 E7 siAN2M1SVP AmAmGfAmGf CfAfCmCmAm AmGmAmAmCm UmAm AS VP-UmsAfsG314 mUmUmCfUmU fGfGmUmGmC mUfCmUfUmG mGmCmUmsUm sGm Conjugate L10- SCmsGmsAmAm 315 E8 siAN1M2SVP GfAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 316 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate L10-S AmsGmsCmCm 317 E9 siAN2M3SVP AmAmGmAmGf CfAfCmCmAm AmGmAmAmCm UmAm ASVP-UmsAfsG 318 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmCmUmsUm sGm ConjugateP10- S CmsCmsAmAm 319 E10 siAN1MASVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 320 mUmUmCfUmU mGmGmUmG mCmUfCmUmG mGmsCmsUm Conjugate R5- SCmsCmsAmAm 321 E11 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 322 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate LA5-S CmsCmsAmAm 323 E12 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 324 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate LB5-S CmsCmsAmAm 325 E13 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 326 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate V8-S CmsCmsAmAm 327 E14 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 328 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate W8-S CmsCmsAmAm 329 E15 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 330 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate X8-S CmsCmsAmAm 331 E16 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 332 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate Z5-S CmsCmsAmAm 333 E17 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 334 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate FIN-S CmsCmsAmAm 335 E18 siAN1M3SVP GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASVP-UmsAfsG 336 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmsCmsUm Conjugate FIN-S AmsGmsCmCm 337 E19 siAN2M3SVP AmAmGmAmGf CfAfCmCmAm AmGmAmAmCm UmAm ASVP-UmsAfsG 338 mUmUmCfUmU mGmGmUmGmC mUfCmUfUmG mGmCmUmsUm sGm ConjugateFIN- S CmsCmsAmAm 339 E20 siAN1M3S GmAmGfCfAf CmCmAmAmGm AmAmCmUmAm ASUmsAfsGmUm 340 UmCfUmUmGm GmUmGmCmUf CmUfUmGmGm sCmsUm Comparative L10-S CmsCmsUmUm 341 Conjugate siHBV GfAmGfGfCf E1 X1M1SVP AmUmAmCmUmUmCmAmAmAm AS VP-UmsUfsU 342 mGmAmAfGmU fAfUmGmCmC mUfCmAfAmG mGmsUmsUmComparative (GalNAc)₃- S AmsCmsAmUm 343 Conjugate 65695 AmUmUfUmGf E2AfUfCmAmGm UmCmUmUmUm UmUm AS AmsAfsAmAm 344 AmGfAmCmUm GmAmUmCmAfAmAfUmAmUm GmUmsUmsGm

TABLE 4F Sequence SEQ Direction: ID Conjugate No 5′-3′ NO  ConjugateL10- S CmsAmsAmUm 345 F1 siAP1M2SVP AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASVP-UmsUfsC 346 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm Conjugate L10-S CmsAmsAmUm 347 F2 siAP1M2SP AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASP-UmsUfsCm 348 UmUmGfUmCm CmAmGmCmUm UfUmAfUmUm GmsGmsGm Conjugate L10-S CmsAmsAmUm 349 F3 siAP1M2SPs AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASPs-UmsUfsC 350 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm Conjugate L10-S CmAmAmUmAm 351 F4 siAP1M2 AmAfGfCfUm GmGmAmCmAm AmGmAmAm AS UmUfCmUmUm352 GfUmCmCmAm GmCmUmUfUm AfUmUmGmGm Gm Conjugate L10- S CmCmCmAmAm 353F5 siAP2M2 UmAmAmAfGf CfUmGmGmAm CmAmAmGmAm Am AS UmUfCmUmUm 354GfUmCmCmAm GmCmUmUfUm AfUmUmGmGm GmAmGm Conjugate L10- S CmAmAmUmAm 355F6 siAP1M2VP AmAfGfCfUm GmGmAmCmAm AmGmAmAm AS VP-UmUfCmU 356 mUmGfUmCmCmAmGmCmUmU fCmAfUmUmG mGmGm Conjugate L10- S CmCmCmAmAm 357 F7 siAP2M2VPUmAmAmAfGf CfUmGmGmAm CmAmAmGmAm Am AS VP-UmUfCmC 358 mUmGfUmCmCmAmGmCmUmU fUmAfUmUmG mGmGmAmGm Conjugate L10- S CmsAmsAmUm 359 F8SiAP1M2S AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm AS UmsUfsCmUm 360 UmGfUmCmCmAmGmCmUmUf UmAfUmUmGm sGmsGm Conjugate L10- S CmsCmsCmAm 361 F9 siAP2M2SAmUmAmAmAf GfCfUmGmGm AmCmAmAmG mAmAm AS UmsUfsCmUm 362 UmGfUmCmCmAmGmCmUmUf UmAfUmUmGm GmGmsAmsGm Conjugate L10- S CmsAmsAmUm 363 F10SiAP1M2SVP AmAmAfGfCf UmGmGmAm CmAmAmGmAm Am AS VP-UmsUfsC 364mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm Conjugate L10- S CmsCmsCmAm365 F11 siAP2M2SVP AmCmAmAmAf GfCfUmGmGm AmCmAmAmGm AmAm AS VP-UmsUfsC366 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmGmGmsAm sGm Conjugate P10- SCmsAmsAmUm 367 F12 siAP1M2SVP AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASVP-UmsUfsC 368 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm Conjugate R5-S CmsAmsAmUm 369 F13 siAP1M2SVP AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASVP-UmsUfsC 370 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm Conjugate LA5-S CmsAmsAmUm 371 F14 siAP1M2SVP AmAmAfGfCf UmGmCmAmCm AmAmGmAmAm ASVP-UmsUfsC 372 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm Conjugate LB5-S CmsAmsAmUm 373 F15 siAP1M2SVP AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASVP-UmsUfsC 374 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm ConjugateV8-siAP1M2SVP S CmsAmsAmUm 375 F16 AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASVP-UmsUfsC 376 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm Conjugate W8-S CmsAmsAmUm 377 F17 siAP1M2SVP AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASVP-UmsUfsC 378 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm ConjugateX8-siAP1M2SVP S CmsAmsAmUm 379 F18 AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASVP-UmsUfsC 380 mUmUmGfUmC mCmAmGmCmU mUfUmAfUmU mGmsGmsGm Conjugate Z5-S CmsAmsAmUm 381 F19 siAP1M2SYP AmAmAfGfCf UmGmGmAmCm AmAmGmAmAm ASVP-UmsUfsCm 382 UmUmGfUmCm CmAmGmCmUm UfUmAfUmUm GmsGmsGm Conjugate FIN-S CmsCmsCmAm 383 F20 siAP2M2S AmUmAmAmAf GfCfUmGmGm AmCmAmAmGm AmAm ASUmsUfsCmUm 384 UmGfUmCmCm AmGmCmUmUf UmAfUmUmGm GmGmsAms Gm ComparativeL10-siHBV S CmsCmsUmUm 385 Conjugate X1M1SVP GfAmGfGfCf F1 AmUmAmCmUmUmCmAmAmAm AS VPUmsUfsUm 386 GmAmAfGmUf AfUmGmCmCm UfCmAfAmGm GmsUmsUmComparative (GalNAc)₃- S GmsCmsUmUm 387 Conjugate 69535 AmAmAmAmGf F2GmGfAmCmAm GmUmAmUmUm CmAm AS UmsGfsAmAm 388 UmAmCmUmGm UmCmCfCmUfUmUmUmAmAm GmCmsAmsAm

TABLE 4G Sequence Direction: SEQ ID Conjugate No 5′-3′ NO Conjugate L10-S GmsAmsAmAm 389 G1 siHB3M1SVP GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASVP-UmsAfsU 390 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Conjugate L10-S GmsAmsAmAm 391 G2 siHB3M1SP GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASP-UmsAfsUm 392 UmCmGfUmUm GmAmCmAmCm AfCmUfUmUm CmsUmsUm Conjugate L10-S GmsAmsAmAm 393 G3 siHB3M1SPs GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASPs-UmsAfsU 394 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Conjugate L10-S GmAmAmAmGm 395 G4 siHB3M1VP UmAfUfGfUm CmAmAmCmGm AmAmUmAm ASVP-UmAfUmU 396 mCmGfUmUmG mAmCmAmUmA fCmUfUmUmC mUmUm Conjugate L10- SGmAmAmAmGm 397 G5 siHB3M1P UmAfUfGfUm CmAmAmCmGm AmAmUmAm AS P-UmAfUmUm398 CmGfUmUmGm AmCmAmUmAf CmUfUmUmCm UmUm Conjugate L10- S GmAmAmAmGm399 G6 siHB3M1 UmAfUfGfUm CmAmAmCmGm AmAmUmAm AS UmAfUmUmCm 400GfUmUmGmAm CmAmUmAfCm UfUmUmCmUm Um Conjugate L10- S GmsAmsAmAm 401 G7siHB3M1S GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm AS UmsAfsUmUm 402 CmGfUmUmGmAmCmAmUmAf CmUfUmUmCm sUmsUm Conjugate L10- S GmsAmsAmAm 403 G8siHB2M1SP GmUmAfUfGfU mCmAmAmCmG mAmAmUmUm AS P-AmsAfsUm 404 UmCmGfUmUmGmAmCmAmUm AfCmUfUmUm CmsCmsAm Conjugate L10- S UmsGmsGmAm 405 G9siHB5M1SVP AmAmGmUmAf UfGfUmCmAm AmCmGmAmAm UmAm AS VP-UmsAfsU 406mUmCmGfUmU mGmAmCmAmU mAf CmUfUmUmCm CmAmsUmsUm Conjugate P10- SGmsAmsAmAm 407 G10 siHB3M1SVP GmUmAfUfGfU mCmAmAmCmG mAmAmUmAm ASVP-UmsAfsU 408 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Conjugate R5-S GmsAmsAmAm 409 G11 siHB3M1SVP GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASVP-UmsAfsU 410 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Conjugate LA5-S GmsAmsAmAm 411 G12 siHB3M1SVP GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASVP-UmsAfsU 412 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Conjugate LB5-S GmsAmsAmAm 413 G13 siHB3M1SVP GmUmAfUfGfUm CmAmAmCmGm AmAmUmAm ASVP-UmsAfsU 414 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Conjugate V8-S GmsAmsAmAm 415 G14 siHB3M1SVP GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASVP-UmsAfsU 416 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Conjugate W8-S GmsAmsAmAm 417 G15 siHB3M1SVP GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASVP-UmsAfsU 418 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Conjugate X8-S GmsAmsAmAm 419 G16 siHB3M1SVP GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASVP-UmsAfsU 420 mUmCmGfUmUm GmAmCmAmUm AfCmUfUmUm CmsUmsUm Conjugate Z5-S GmsAmsAmAm 421 G17 siHB3M1SVP GmUmAfUfGf UmCmAmAmCm GmAmAmUmAm ASVP-UmsAfsU 422 mUmCmGfUmU mGmAmCmAmU mAfCmUfUmU mCmsUmsUm Comparative NCS UUCUCCGAAC 423 Conjugate GUGUCACGU G1 AS ACGUGACACG 424 UUCGGAGAAU U*S: sense strand; AS: antisense strand Note: C, G, U, and A representsthe base components of the nucleotides; m represents that the nucleotideadjacent to the left side of the letter m is a 2′-methoxy modifiednucleotide; f represents that the nucleotide adjacent to the left sideof the letter f is a 2′-fluoro modified nucleotide; s represents thatthe two nucleotides adjacent to both sides of the letter s are linked bya phosphorothioate linkage; VP represents that the nucleotide adjacentto the right side of the letter VP is a vinyl phosphate modifiednucleotide; P represents that the nucleotide adjacent to the right sideof the letter P is a phosphate nucleotide; Ps represents that thenucleotide adjacent to the right side of the letters Ps is aphosphorothioate modified nucleotide.

In the following Preparation Examples 4-12, various conjugatingmolecules were synthesized and were respectively used to replaceCompound L-10 in Preparation Example 2; and it is expected thatConjugates A12-A19, B8-B15, C4-C11, D4-D11, E10-E17, F12-F19, andG10-G17 listed in Tables 4A-4G can be prepared based on thecorresponding sequences listed in Tables 4A-4G.

Preparation Example 4 Preparation of P10 Conjugates

In this Preparation Example, it is expected that Conjugates A12, B8, C4,D4, E10, F12 and G10 (hereinafter referred to as P-10 Conjugates) can besynthesized according to the following process.

(4-1) Synthesis of P-10 Compounds

P-10 Compounds were synthesized according to the following process:

(4-1-1) Synthesis of GAL5-C4-1

GAL-5 (13.43 g, 30.0 mmol) obtained according to the method described instep (2-1-1) above, t-butyl 4-aminobutyrate hydrochloride (5.87 g, 30.0mmol), O-benzotriazol-tetramethyluronium hexafluorophosphate (13.65 g,36.0 mmol) and diisopropylethylamine (11.63 g, 90.0 mmol) were addedinto 40 ml of N,N-dimethylformamide, dissolved homogeneously and thenstirred at room temperature to react for 5 hours. The resultant reactionsolution was added with 300 ml of saturated aqueous sodium bicarbonatesolution, extracted three times, each with 200 ml of ethyl acetate. Allorganic phases were combined and washed once with 200 ml of saturatedbrine. The organic phase was isolated and dried with anhydrous sodiumsulfate. The solvent was removed by evaporation under reduced pressureto dryness to give 30.3 g of crude product GAL5-C4-1 as oil, which wasdirectly used in the next reaction.

(4-1-2) Synthesis of GAL5-C4-2

The crude product GAL5-C4-1 (30.3 g, 30 mmol) obtained in step (4-1-1)was dissolved in 180 ml of formic acid and stirred at room temperatureto react for 16 hours. The solvent was evaporated to dryness. Theresidue was purified by column chromatography (200-300 mesh normal phasesilica gel, with a gradient elution ofdichloromethane:methanol=100:18-100:20). The eluate was collected andconcentrated to remove the solvents to give a total of 14.84 g of thetarget product GAL5-C4-2.

(4-1-3) Synthesis of P-6:

M-18-Tr (2.02 g, 4.69 mmol) obtained according to the method describedin step (2-1-4) and GAL5-C4-2 (8.24 g, 15.48 mmol) obtained in step(4-1-2) were mixed and dissolved in 47 ml of acetonitrile, added withN-methylmorpholine (3.13 g, 30.96 mmol) followed by4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM,4.28 g, 15.48 mmol) to react under stirring at room temperature for 2hours. The resultant reaction solution was diluted with 20 ml ofdichloromethane. The resultant organic phase was washed with 10 ml ofsaturated sodium bicarbonate solution and 10 ml of saturated brine,respectively. All organic phases were combined, dried with anhydroussodium sulfate, and filtered. The solvent was removed by evaporationunder reduced pressure to give a crude product, which was purified byusing a normal phase silica gel column (200-300 mesh). The column waspacked with petroleum ether, added with 1 wt % triethylamine forneutralizing the acidity of silica gel, and eluted with a gradientelution of dichloromethane:methanol=100:5-100:7. The eluate wascollected, and the solvent was removed by evaporation under reducedpressure to give a total of 8.27 g of pure product P-6.

(4-1-4) Synthesis of P-7:

P-6 (6.82 g, 3.456 mmol) obtained in step (4-1-3) above was dissolved in69 ml of dichloromethane, and added with dichloroacetic acid (13.367 g,103.67 mmol) to react at room temperature for 2 hours. The resultantreaction solution was diluted by adding 100 ml of dichloromethane,washed and adjusted to pH 7-8 with saturated sodium bicarbonatesolution. The aqueous phase isolated was extracted six times, each with30 ml of dichloromethane. All organic phases were combined, dried withanhydrous sodium sulfate, and filtered. Then the solvent was removed byevaporation under reduced pressure to give a crude product. The crudeproduct was purified by using a normal phase silica gel column (200-300mesh). The column was added with 10 wt % triethylamine for neutralizingthe acidity of silica gel, equilibrated with 1 wt ‰ triethylamine andeluted with a gradient elution ofdichloromethane:methanol=100:30-100:40. The eluate was collected, andthe solvent was removed by evaporation under reduced pressure to give atotal of 4.82 g of P-7. MS m/z: C78H127N10O33, [M+H]+, calculated:1732.91, measured: 1735.73.

(4-1-5) Synthesis of P-8:

P-7 (2.653 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed anddissolved in 16 ml of dichloromethane, and added with3-diethoxyphosphoryl-1,2,3-benzotrizin 4(3H)-one (DEPBT) (1.375 g, 4.596mmol) followed by diisopropylethylamine (1.188 g, 9.191 mmol) to reactunder stirring at 25° C. for 2 hours. The organic phase was washed with10 ml of saturated sodium bicarbonate. The aqueous phase isolated wasextracted three times, each with 10 ml of dichloromethane. All organicphases were combined and washed with 10 ml of saturated brine. Theaqueous phase isolated was extracted twice, each with 10 ml ofdichloromethane, and the obtained organic phases were combined, driedwith anhydrous sodium sulfate and filtered. The solvent was removed byevaporation under reduced pressure, and foam-dried in a vacuum oil pumpovernight to give a crude product. The crude product was subjected to acolumn purification. The column was filled with 120 g normal phasesilica gel (200-300 mesh), added with 20 ml triethylamine forneutralizing the acidity of silica gel, equilibrated with petroleumether containing 1 wt % triethylamine and eluted with a gradient elutionof petroleum ether:ethylacetate:dichloromethane:N,N-dimethylformamide=1:1:1:0.5-1:1:1:0.6. Theeluate was collected, and the solvent was removed by evaporation underreduced pressure to give a total of 2.793 g of pure product P-8.

(4-1-6) Synthesis of P-9:

P-8 (490 mg, 0.231 mmol), succinic anhydride (69 mg, 0.693 mmol) and4-dimethylaminopyridine (DMAP, 68 mg, 0.554 mmol) were mixed anddissolved in 2.3 ml of dichloromethane, and added withdiisopropylethylamine (DIEA, 149 mg, 1.155 mmol) to react under stirringat 25° C. for 21 hours. The resultant reaction solution was added with50 ml dichloromethane for dilution and then washed with 100 ml of 0.5 Mtriethylamine phosphate. The aqueous phase isolated was extracted threetimes, each with 10 ml of dichloromethane. All organic phases werecombined, and the solvent was removed by evaporation under reducedpressure to give a crude product. The crude product was subjected to acolumn purification. The column was filled with 80 g normal phase silicagel (200-300 mesh), added with 1 wt % triethylamine for neutralizing theacidity of silica gel and equilibrated with dichloromethane, and elutedwith a gradient elution of dichloromethane containing 1 wt ‰triethylamine:methanol=100:18-100:20. The eluate was collected, and thesolvent was removed by evaporation under reduced pressure to give atotal of 200 mg of pure product P-9 conjugating molecule. MS m/z:C106H153N10O41, [M-DMTr]+, calculated: 1921.05, measured: 1920.97.

(4-1-7) Synthesis of P-10:

P-10 was prepared by using the same method as that in step (2-1-9) ofPreparation Example 2, except that: P-9 conjugating molecule was used toreplace L-9 conjugating molecule, thereby obtaining P-9 conjugatingmolecule linked to a solid phase support.

(4-2) Synthesis of P10 Conjugates

Conjugates were prepared by using the same methods as those in steps(2-2), (2-3A) and (2-4) of Preparation Example 2, except that P-10Compound was used to replace L-10 Compound to start the synthesis of asense strand. It is expected that Conjugates A12, B8, C₄, D4, E10, F12and G10 with a structure as shown by Formula (404) can be obtained.

Preparation Example 5 Preparation of R5 Conjugates

In this Preparation Example, Conjugates A13, B9, C5, D5, E11, F13 andG11 (hereinafter referred to as R5 Conjugates) can be synthesized by thefollowing method.

(5-1) Synthesis of R-5 Compound

R-5 Compound was synthesized by the following method:

(5-1-1) Synthesis of GAL-C7-1

GAL-3 (26.4 g, 80.2 mmol) obtained according to the method described instep (2-1-Tb) was dissolved in 134 ml of anhydrous 1,2-dichloroethane,and added with 60 g of 4 Å molecular sieve powder followed by7-octen-1-ol (11.3 g, 88.2 mmol) to react under stirring at roomtemperature for 10 minutes. Trimethylsilyl trifluoromethanesulphonate(8.9 g, 40.1 mmol) was added in an ice bath and nitrogen atmosphere toreact under stirring at room temperature for 24 hours. The 4 Å molecularsieve powder was removed by filtration. 500 ml of saturated aqueoussodium bicarbonate solution was added to the filtrate for washing. Anorganic phase was isolated. The aqueous phase was extracted once with100 ml of dichloromethane. All organic phases were combined and washedonce with 250 ml of saturated brine. The organic phase was isolated anddried with anhydrous sodium sulfate. The solvent was removed byevaporation under reduced pressure to dryness to give 33.3 g of productGAL-C7-1 as a yellow syrup, which was directly used in the nextoxidation reaction without purification.

(5-1-2) Synthesis of GAL-C7-2

GAL-C7-1 (33.3 g, 72.8 mmol) obtained in step (5-1-1) was dissolved in amixed solvent of 160 ml of dichloromethane and 160 ml of acetonitrile,added with 216 ml of water and solid sodium periodate (62.3 g, 291.2mmol) respectively, stirred in an ice water bath for 10 minutes, andadded with a catalyst ruthenium trichloride (498 mg, 2.4 mmol). Thereaction was naturally warmed to room temperature and stirred for 23hours. The resultant reaction solution was diluted by adding 200 ml ofwater under stirring, and adjusted to pH 7.5 by adding saturated sodiumbicarbonate. The organic phase was isolated and discarded. The aqueousphase was extracted three times, each with dichloromethane. The organicphases resulted from the extraction were discarded. The aqueous phaseresulted from the extraction was adjusted to a pH of about 3 with citricacid solid and extracted three times, each with 200 ml ofdichloromethane, and the resultant organic phases were combined anddried with anhydrous sodium sulfate. The solvent was removed byevaporation under reduced pressure, and then the residue was purified bycolumn chromatography (200-300 mesh normal phase silica gel, with agradient elution of dichloromethane:methanol=100:18-100:20) to give 22.4g of product GAL-C7-2 as a white foamy solid. MS m/z: C21H32NO11,[M+H]+, calculated: 476.50, measured: 475.94.

(5-1-3) Synthesis of R-1:

M-18-Tr (2.02 g, 4.69 mmol) obtained according to the method describedin step (2-1-4) and GAL-C7-2 (7.36 g, 15.48 mmol) were mixed anddissolved in 47 ml of acetonitrile, added with N-methylmorpholine (3.13g, 30.96 mmol) followed by4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM,4.28 g, 15.48 mmol) to react under stirring at room temperature for 2hours. The resultant reaction solution was diluted with 200 ml ofdichloromethane. The organic phase was washed with 100 ml of saturatedsodium bicarbonate solution and 100 ml of saturated brine, respectively.All organic phases were combined, dried with anhydrous sodium sulfate,and filtered. The solvent was removed by evaporation under reducedpressure to give a crude product, which was purified by using a normalphase silica gel column (200-300 mesh). The column was packed withpetroleum ether, added with 1 wt % triethylamine for neutralizing theacidity of silica gel, and eluted with a gradient elution ofdichloromethane:methanol=100:5-100:7. The eluate was collected and thesolvent was removed by evaporation under reduced pressure to give 7.82 gof pure product R-1.

(5-1-4) Synthesis of R-2:

R-1 (6.23 g, 3.456 mmol) was dissolved in 69 ml of dichloromethane, andadded with dichloroacetic acid (13.367 g, 103.67 mmol) to react at roomtemperature for 2 hours. The resultant reaction solution was diluted byadding 100 ml of dichloromethane, washed and adjust to pH 7-8 withsaturated sodium bicarbonate solution. The aqueous phase isolated wasextracted six times, each with 30 ml of dichloromethane. All organicphases were combined, dried with anhydrous sodium sulfate, and filtered.Then the solvent was removed by evaporation under reduced pressure togive a crude product. The crude product was purified by using a normalphase silica gel column (200-300 mesh). The column was added with 10 wt% triethylamine for neutralizing the acidity of silica gel andequilibrated with 1 wt ‰ triethylamine, and eluted with a gradientelution of dichloromethane:methanol=100:30-100:40. The solvent wasremoved by evaporation under reduced pressure to give 4.49 g of pureproduct R-2.

(5-1-5) Synthesis of R-3:

R-2 (2.391 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed anddissolved in 16 ml of dichloromethane, and added with3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one (DEPBT, 1.375 g,4.596 mmol) followed by diisopropylethylamine (1.188 g, 9.191 mmol) toreact under stirring at 25° C. for 2 hours The organic phase was washedwith 10 ml of saturated sodium bicarbonate. The aqueous phase isolatedwas extracted three times, each with 10 ml of dichloromethane. Theorganic phase was washed with 10 ml of saturated brine. The aqueousphase isolated was extracted twice, each with 10 ml of dichloromethane,and the obtained organic phases were combined, dried with anhydroussodium sulfate and filtered. The solvent was removed by evaporationunder reduced pressure, and the residue was foam-dried in a vacuum oilpump overnight to give a crude product. The crude product was subjectedto a column purification. The column was filled with 120 g normal phasesilica gel (200-300 mesh), added with 20 ml triethylamine forneutralizing the acidity of silica gel and equilibrated with petroleumether containing 1 wt % triethylamine, and eluted with a gradientelution of petroleum ether:ethylacetate:dichloromethane:N,N-dimethylformamide=1:1:1:0.5-1:1:1:0.6. Thesolvent was removed by evaporation under reduced pressure to give 2.642g of pure product R-3.

(5-1-6) Synthesis of R-4:

R-3 (795 mg, 0.4074 mmol), succinic anhydride (82 mg, 0.8148 mmol) and4-dimethylaminopyridine (DMAP, 100 mg, 0.8148 mmol) were mixed anddissolved in 4 ml of dichloromethane, and added withdiisopropylethylamine (DIEA, 100 mg, 0.8148 mmol) to react understirring at 25° C. for 18 hours. The resultant reaction solution waswashed with 5 ml of 0.5 M triethylamine phosphate. The aqueous phase wasextracted three times, each with 5 ml of dichloromethane. All organicphases were combined, and the solvent was removed by evaporation underreduced pressure to give a crude product. The crude product wassubjected to a column purification. The column was filled with 30 gnormal phase silica gel (200-300 mesh), added with 1 wt % triethylaminefor neutralizing the acidity of silica gel and equilibrated withdichloromethane, and eluted with a gradient elution of dichloromethanecontaining 1 wt ‰ triethylamine:methanol=100:18-100:20. The eluate wascollected, and the solvent was removed by evaporation under reducedpressure to give 505 mg of pure product of R-4 conjugating molecule.

(5-1-7) Synthesis of R-5 Conjugating Molecule

R-5 was prepared by using the same method as that in step (2-1-9) ofPreparation Example 2, except that: R-4 conjugating molecule was used toreplace L-9 conjugating molecule, thereby obtaining R-4 conjugatingmolecule linked to a solid phase support.

(5-2) Synthesis of R5 Conjugates

R5 Conjugates were prepared by using the same methods as those in steps(2-2), (2-3A) and (2-4) of Preparation Example 2, except that R-5Compound was used to replace L-10 Compound to start the synthesis of asense strand. It is expected that Conjugates A13, B9, C5, D5, E11, F13and G11 with a structure as shown by Formula (407) can be obtained.

Preparation Example 6 Preparation of LA-5 Conjugates

In this Preparation Example, Conjugates A14, B10, C6, D6, E12, F14 andG12 (hereinafter referred to as LA-5 Conjugates) can be synthesized bythe following method.

It is expected that LA-5 Compounds can be synthesized by the followingprocess route:

LA Conjugates were prepared by using the same methods as those in steps(2-2), (2-3A) and (2-4) of Preparation Example 2, except that LA-5Compound was used to replace L-10 Compound to start the synthesis of asense strand. It is expected that Conjugates A14, B10, C6, D6, E12, F14and G12 with a structure as shown by Formula (412) can be obtained.

Preparation Example 7 Preparation of LB-5 Conjugates

In this Preparation Example, Conjugates A15, B11, C7, D7, E13, F15 andG13 (hereinafter referred to as LB-5 Conjugates) can be synthesized bythe following method.

(7-1) Synthesis of LB-5 Compounds

LB-5 Compounds were synthesized by the following method:

(7-1-1) Synthesis of LB-1

L-8 (5.0 g, 3.386 mmol) obtained according to the method described instep (2-1-6), adipic anhydride (870 mg, 6.772 mmol) and4-dimethylaminopyridine (DMAP, 827 mg, 6.772 mmol) were mixed anddissolved in 130 ml of dichloromethane, and added withdiisopropylethylamine (DIEA, 2.2 g, 16.931 mmol) to react under stirringat 25° C. for 4 hours. The resultant reaction solution was added with 70ml dichloromethane for dilution and then washed with 0.5 M triethylaminephosphate. The aqueous phase isolated was extracted four times, eachwith 10 ml of dichloromethane. All organic phases were combined, and thesolvent was removed by evaporation under reduced pressure to give acrude product. The crude product was subjected to a column purification.The column was filled with 120 g normal phase silica gel (200-300 mesh),added with 1 wt % triethylamine for neutralizing the acidity of silicagel and equilibrated with dichloromethane, and eluted with a gradientelution of petroleum ether:ethylacetate:dichloromethane:methanol=1:1:1:0.2-1:1:1:1. The solvent wasremoved by evaporation under reduced pressure to give 4.267 g of pureproduct LB-1.

(7-1-2) Synthesis of LB-2:

LB-1 (4.697 g, 2.753 mmol, combination of 2 batches) obtained accordingto the method described in step (7-1-1), 3-amino-1,2-propanediol (313mg, 3.442 mmol), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinehydrochloride (DMTMM, 953 mg, 3.442 mmol) and N-methylmorpholine (700mg, 6.884 mmol) were sequentially added to the mixture of 30 ml ofacetonitrile and 3 ml of methanol to react under stirring at roomtemperature overnight. The solvent was evaporated to dryness, and theresidue was purified by column chromatography (200-300 mesh normal phasesilica gel, with a gradient elution ofdichloromethane:methanol=1:0.07-1:0.5). The eluate was collected andconcentrated to remove the solvents to give 3.27 g of target productLB-2.

(7-1-3) Synthesis of LB-3:

LB-2 (2.27 g, 1.353 mmol) was dissolved in 14 ml of anhydrous pyridine,and added with 4,4′-dimethoxytrityl chloride (688 mg, 2.03 mmol) toreact under stirring at room temperature overnight. The reaction wasquenched by addition of 150 ml of methanol. The solvent was evaporatedto dryness, and the residue was purified by column chromatography(200-300 mesh normal phase silica gel, with a gradient elution ofdichloromethane:methanol=1:0.05-1:0.2). The eluate was collected andconcentrated to remove the solvent to give 1.647 g of target productLB-3.

(7-1-4) Synthesis of LB-4:

LB-3 (822 mg, 0.415 mmol), succinic anhydride (83 g, 0.83 mmol) and4-dimethylaminopyridine (DMAP, 102 mg, 0.83 mmol) were mixed anddissolved in 4 ml of dichloromethane, added with DIEA (270 mg, 2.075mmol), and stirred at 25° C. to react overnight. The resultant reactionsolution was washed with 0.5 M triethylamine phosphate three times. Theaqueous phase isolated was extracted three times, each with 2 ml ofdichloromethane. All organic phases were combined, and the solvent wasremoved by evaporation under reduced pressure to give a crude product.The crude product was subjected to a column purification. The column wasfilled with normal phase silica gel (200-300 mesh), added with 5 wt %triethylamine for neutralizing the acidity of silica gel andequilibrated with petroleum ether, and eluted with a gradient elution of1 wt ‰ triethylamine-containing dichloromethane:methanol=100:5-100:20.The solvent was removed by evaporation under reduced pressure to give787 mg of pure product LB-4 conjugating molecule.

(7-1-5) Synthesis of LB-5:

LB-5 was prepared by using the same method as that in step (2-1-9) ofPreparation Example 2, except that: LB-4 conjugating molecule was usedto replace L-9 conjugating molecule, thereby obtaining LB-4 conjugatingmolecule linked to a solid phase support.

(7-2) Synthesis of LB-5 Conjugates

LB-5 Conjugates were prepared by using the same methods as those insteps (2-2), (2-3A) and (2-4) of Preparation Example 2, except that LB-5Compound was used to replace L-10 compound to start the synthesis of asense strand. It is expected that Conjugates A15, B11, C7, D7, E13, F15and G13 with a structure as shown by Formula (413) can be obtained.

Preparation Example 8, Synthesis of V8 Conjugates

In this Preparation Example, it is expected that Conjugates A16, B12,C8, D8, E14, F16 and G14 (hereinafter referred to as V8 Conjugates) canbe synthesized by the following method.

It is expected that V-8 Compounds can be synthesized by the followingprocess route:

V8 Conjugates were prepared by using the same methods as those in steps(2-2), (2-3A) and (2-4) of Preparation Example 2, except that V-8Compound was used to replace L-10 Compound to start the synthesis of asense strand. It is expected that Conjugates A15, B12, C8, D8, E14, F16and G14 (hereinafter referred to as V8 Conjugates) with a structure asshown by Formula (414) can be obtained.

Preparation Example 9 Synthesis of W8 Conjugates

In this Preparation Example, it is expected that Conjugates A17, B13,C9, D9, E15, F17 and G15 (hereinafter referred to as W8 Conjugates) canbe synthesized by the following method.

(9-1) Synthesis of W-8 Compounds

W-8 Compounds were synthesized by the following method:

(9-1-1) Synthesis of W-1

W-0 (202 g, 10 mmol) was dissolved in 25 ml of acetonitrile, added withtriethylamine (4.048 g, 40 mmol), and cooled to about 0° C. in an icewater bath. Ethyl trifluoroacetate (5.683 g, 40 mmol) was added to reactat room temperature for 22 hours. The solvent was removed by evaporationunder reduced pressure, and the residue was foam-dried in a vacuum oilpump for 18 hours to give 5.835 g of crude solid product W-1.

(9-1-2) Synthesis of W-2:

The crude product W-1 (5.835 g, 10 mmol) was dissolved in 50 ml ofdichloromethane. The resultant reaction solution was added with TrCl(3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) to react understirring at room temperature for 20 hours. The resultant reactionsolution was washed twice, each with 20 ml of saturated sodiumbicarbonate and once with 20 ml of saturated brine. All organic phaseswere combined, dried with anhydrous sodium sulfate and filtered. Theorganic solvent was removed by evaporation under reduced pressure, andthe residue was foam-dried in a vacuum oil pump overnight to give 8.012g of crude solid product W-2. The crude solid product W-2 was used inthe next deprotection reaction without treatment.

(9-1-3) Synthesis of W-3:

The crude product W-2 (8.012 g, 10 mmol) was dissolved in 100 ml ofmethanol, and added with 100 ml of aqueous methylamine solution (40 wt%) to react under stirring at 50° C. for 23 hours. Insoluble particleswere removed by filtration. The solvent was removed by evaporation underreduced pressure. The residue was added with 200 ml of mixed solvent ofDCM:methanol in a volume ratio of 1:1, and the resultant organic phasewas washed with 50 ml of saturated sodium bicarbonate. The aqueous phaseisolated was extracted three times, each with 50 ml of dichloromethane.All organic phases were combined, dried with anhydrous sodium sulfateand filtered. The solvent was removed by evaporation under reducedpressure, and the residue was foam-dried in a vacuum oil pump overnight,and purified by using a normal phase silica gel column (200-300 mesh).The column was packed with petroleum ether, added with 1 wt %triethylamine for neutralizing the acidity of silica gel, and elutedwith a gradient elution of dichloromethane:methanol:aqueous ammonia (25wt %)=1:1:0.05-1:1:0.25. The eluate was collected. The solvent wasremoved by evaporation under reduced pressure, and the residue wasfoam-dried in a vacuum oil pump to give 3.062 g of pure product W-3.

(9-1-4) Synthesis of W-4:

W-3 (0.675 g, 1.517 mmol) and GAL-C7-2 (2.60 g, 5.46 mmol) were mixedand dissolved in 47 ml of acetonitrile, added with diisopropylethylamine(1.57 g, 12.14 mmol) followed by3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one (DEPBT, 1.816 g,6.04 mmol) to react under stirring at room temperature for 2.5 hours.The resultant reaction solution was diluted with 100 ml ofdichloromethane. The organic phase obtained was washed with 80 ml ofsaturated sodium bicarbonate solution and 80 ml of saturated brine,respectively. All organic phases were combined, dried with anhydroussodium sulfate, and filtered. The solvent was removed by evaporationunder reduced pressure to give a crude product, which was purified byusing a normal phase silica gel column (200-300 mesh). The column waspacked with petroleum ether, added with 1 wt % triethylamine forneutralizing the acidity of silica gel, and eluted with a gradientelution of dichloromethane:methanol=100:5-100:7. The eluate wascollected, and the solvent was removed by evaporation under reducedpressure to give 1.610 g of pure product W-4.

(9-1-5) Synthesis of W-5:

W-4 (1.61 g, 0.886 mmol) was dissolved in 125 ml of dichloromethane, andadded with dichloroacetic acid (3.5 ml, 42.43 mmol) to react at roomtemperature for 1 hour. The resultant reaction solution was neutralizedby adding 150 ml of pyridine. The solvent was removed by evaporationunder reduced pressure to give a crude product. The crude product waspurified by using a normal phase silica gel column (200-300 mesh). Thecolumn was added with 10 wt % triethylamine for neutralizing the acidityof silica gel, equilibrated with 1 wt ‰ triethylamine and eluted with agradient elution of dichloromethane:methanol=100:30-100:40. The eluatewas collected, and the solvent was removed by evaporation under reducedpressure to give 1.26 g of pure product W-5.

(9-1-6) Synthesis of W-6:

W-5 (1.25 g, 0.793 mmol) and A-1 (1.21 g, 2.38 mmol) obtained accordingto the method described in step (2-1-7a) were mixed and dissolved in 12ml of dichloromethane, and added with3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one (DEPBT, 0.712 g,2.38 mmol) followed by diisopropylethylamine (0.615 g, 4.76 mmol) toreact under stirring at 25° C. for 3 hours. The organic phase was washedwith 80 ml of saturated sodium bicarbonate. The aqueous phase isolatedwas extracted three times, each with 10 ml of dichloromethane. Allorganic phases were combined and washed with 10 ml of saturated brine.The obtained organic phases were combined, dried with anhydrous sodiumsulfate and filtered. The solvent was removed by evaporation underreduced pressure, and the residue was foam-dried in a vacuum oil pumpovernight to give a crude product. The crude product was subjected to acolumn purification. The column was filled with 185 g normal phasesilica gel (200-300 mesh), added with 20 ml triethylamine forneutralizing the acidity of silica gel, equilibrated with petroleumether containing 1 wt % triethylamine and eluted with a gradient elutionof petroleum ether:ethylacetate:dichloromethane:N,N-dimethylformamide=1:1:1:0.1-1:1:1:0.7. Theeluate was collected, and the solvent was removed by evaporation underreduced pressure to give 1.57 g of pure product W-6.

(9-1-7) Synthesis of W-7:

W-6 (1.238 g, 0.63 mmol), succinic anhydride (0.189 g, 1.89 mmol) and4-dimethylaminopyridine (DMAP, 0.231 g, 1.89 mmol) were mixed anddissolved in 7 ml of dichloromethane, and added with DIEA (0.407 g, 3.15mmol) to react under stirring at 25° C. for 24 hours. The resultantreaction solution was washed with 5 ml of 0.5 M triethylamine phosphate.The aqueous phase isolated was extracted three times, each with 5 ml ofdichloromethane. All organic phases were combined, and the solvent wasremoved by evaporation under reduced pressure to give a crude product.The crude product was subjected to a column purification. The column wasfilled with 30 g normal phase silica gel (200-300 mesh), added with 1 wt% triethylamine for neutralizing the acidity of silica gel, equilibratedwith dichloromethane and eluted with a gradient elution of 1 wt ‰triethylamine-containing dichloromethane:methanol=100:18-100:20. Theeluate was collected, and the solvent was removed by evaporation underreduced pressure to give 1.033 g of pure product W-7 conjugatingmolecule. MS m/z: C101H146N7O38, [M-DMTr]+, calculated: 1763.92,measured: 1763.21.

(9-1-8) Synthesis of W-8

W-8 was prepared by using the same method as that in step (2-1-9) ofPreparation Example 2, except that: W-7 conjugating molecule was used toreplace L-9 conjugating molecule, thereby obtaining W-7 conjugatingmolecule linked to a solid phase support.

(9-2) Synthesis of W-8 Conjugates

W-8 Conjugates were prepared by using the same methods as those in steps(2-2), (2-3A) and (2-4) of Preparation Example 2, except that W-8Compound was used to replace L-10 Compound to start the synthesis of asense strand. It is expected that Conjugates A17, B13, C9, D9, E15, F17and G15 with a structure as shown by Formula (415) can be obtained.

Preparation Example 10 Synthesis of X8 Conjugates

In this Preparation Example, it is expected that Conjugates A18, B14,C10, D10, E16, F18 and G16 (hereinafter referred to as X8 Conjugates)can be synthesized by the following method.

It is expected that X-8 Compounds can be synthesized by the followingprocess route:

X-8 Conjugates were prepared by using the same methods as those in steps(2-2), (2-3A) and (2-4) of Preparation Example 2, except that X-8Compound was used to replace L-10 Compound to start the synthesis of asense strand. It is expected that Conjugates A18, B14, C10, D10, E16,F18 and G16 with a structure as shown by Formula (421) can be obtained.

Preparation Example 11 Synthesis of Z-5 Conjugates

In this Preparation Example, it is expected that Conjugates A19, B15,C11, D11, E12, F14 and G12 (hereinafter referred to as Z5 Conjugates)can be synthesized by the following method.

(11-1) Synthesis of Z-5 Compounds

Z-5 Compounds can be synthesized by the following method:

(11-1-1) Synthesis of Z-1

W-3 (1.50 g, 3.37 mmol) obtained according to the method described instep (9-1-3) and GAL5-C4-2 (7.18 g, 13.48 mmol) obtained according tothe method described in step (4-1-2) were mixed and dissolved in 34 mlof dichloromethane, and added with diisopropylethylamine (3.48 g, 26.96mmol) followed by 3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one(DEPBT, 4.04 g, 13.48 mmol) to react under stirring at room temperaturefor 4.5 hours. The resultant liquid solution was diluted with 100 ml ofdichloromethane. The organic phase was washed with 80 ml of saturatedsodium bicarbonate solution and 80 ml of saturated brine, respectively.All organic phases were combined, dried with anhydrous sodium sulfate,and filtered. The solvent was removed by evaporation under reducedpressure to give a crude product, which was purified by using a normalphase silica gel column (200-300 mesh). The column was packed withpetroleum ether, added with 1 wt % triethylamine for neutralizing theacidity of silica gel, and eluted with a gradient elution ofdichloromethane:methanol=30:1-15:1. The eluate was collected and removedby evaporation under reduced pressure to give 3.97 g of pure productZ-1. MS m/z: C98H143N10O33, [M+H]+, calculated: 1987.98, measured:1987.90.

(11-1-2) Synthesis of Z-2:

Z-1 (3.97 g, 2.00 mmol) was dissolved in 250 ml of dichloromethane, andadded with dichloroacetic acid (10.941 g, 84.85 mmol) to react at roomtemperature for 1 hour. Pyridine was added to neutralize the resultantreaction solution to neutral. The solvent was removed by evaporationunder reduced pressure to give a crude product. The column was loadedwith 200 g 200-300 mesh normal phase silica gel, and added with 10 wt %pyridine for neutralizing the acidity of silica gel, equilibrated with 1wt ‰ pyridine and eluted with a gradient elution ofdichloromethane:methanol=10:1-2:1. The eluate was collected, and thesolvent was removed by evaporation under reduced pressure to give 3.49 gof pure product Z-2. MS m/z: C79H129N10O33, [M+H]+, calculated: 1746.94,measured: 1746.90.

(11-1-3) Synthesis of Z-3:

Z-2 (3.49 g, 2.0 mmol) and A-1 (3.06 g, 6.0 mmol) obtained according tothe method described in step (2-1-7a) were mixed and dissolved in 30 mlof dichloromethane, and added with3-(diethoxyphosphoryloxy)-1,2,3-benzotrizin-4(3H)-one (DEPBT, 1.80 g,6.0 mmol) followed by diisopropylethylamine (1.55 g, 12.0 mmol) to reactfor 3 hours under stirring at 25° C. The resultant reaction solution wasadded with 100 ml dichloromethane for dilution. The organic phase waswashed twice with 30 ml of saturated sodium bicarbonate. The aqueousphase was extracted with 10 ml of dichloromethane. All organic phaseswere combined and washed with 50 ml of saturated brine. The obtainedorganic phases were combined and dried with anhydrous sodium sulfate,and filtered. The solvent was removed by evaporation under reducedpressure, and the residue was foam-dried in a vacuum oil pump overnightto give a crude product. The crude product was subjected to a columnpurification. The column was filled with 200 g normal phase silica gel(200-300 mesh), added with 20 ml triethylamine for neutralizing theacidity of silica gel. The column was equilibrated with petroleum ethercontaining 1 wt % triethylamine and eluted with a gradient elution ofdichloromethane:methanol=25:1-15:1. The eluate was collected, and thesolvent was removed by evaporation under reduced pressure to give 2.2 gof pure product Z-3. MS m/z: C103H151N10O38, [M+H]+, calculated:2136.02, measured: 2136.20.

(11-1-4) Synthesis of Z-4:

Z-3 (2.10 g, 0.983 mmol) was dissolved in 14.8 ml of dichloromethanecontaining DIEA (0.635 g, 4.915 mmol), 4-dimethylaminopyridin (DMAP, 240mg, 1.966 mmol) was added to the resultant solution and stirred untilthe solution is clear. Succinic anhydride (197 mg, 1.966 mmol) was addedto react under stirring at 25° C. for 18 hours. The resultant reactionsolution was added with 50 ml dichloromethane for dilution, and washedwith 80 ml of 0.5 M triethylamine phosphate. The aqueous phase wasextracted twice, each with 50 ml of dichloromethane. All organic phaseswere combined, and the solvent was removed by evaporation under reducedpressure to give a crude product. The crude product was subjected to acolumn purification. The column was filled with 188 g normal phasesilica gel (200-300 mesh), added with 1 wt % triethylamine forneutralizing the acidity of silica gel, equilibrated withdichloromethane and eluted with a gradient elution of dichloromethanecontaining 1 wt ‰ triethylamine:methanol=10:1-3:1. The eluate wascollected, and the solvent was removed by evaporation under reducedpressure to give 1.95 g of pure product Z-4 conjugating molecule. MSm/z: C107H155N10O41, [M+H]+, calculated: 1935.07, measured: 1935.29.

(11-1-5) Synthesis of Z-5

Z-5 was prepared by using the same method as that in step (2-1-9) ofPreparation Example 2, except that: Z-4 conjugating molecule was used toreplace L-9 conjugating molecule, thereby obtaining Z-4 conjugatingmolecule linked to a solid phase support.

(11-2) Synthesis of Z-5 Conjugates

Z-5 Conjugates were prepared by using the same methods as those in steps(2-2), (2-3A) and (2-4) of Preparation Example 2, except that Z-5Compound was used to replace L-10 Compound to start the synthesis of asense strand. It is expected that Conjugates A19, B15, C11, D11, E17,F19 and G17 with a structure as shown by Formula (422) can be obtained.

Preparation Example 12 Preparation of FIN Conjugates

In this Preparation Example, Conjugates A20-A23, B16-B17, C14, D14,E18-E20, F20 and Comparative Conjugates A1 and B1 (hereinafter referredto as FIN Conjugates) listed in Tables 4A-4G were synthesized. For thesequences of the conjugated siRNA in these conjugates, please refer tothe corresponding sequences listed in Tables 4A-4G.

(12-1) Synthesis of FIN-2 conjugating molecule

FIN-2 conjugating molecule was synthesized with reference to thepreparation method described in Rajeev et al., ChemBioChem 2015, 16,903-908 according to the following process route:

(12-1-1) Synthesis of compound PRO-10

(12-1-1a) Synthesis of PRO-7

2.93 g of PRO-6 (L-hydroxyproline, CAS No.: 51-35-4, purchased fromEnergy Chemical, 22.4 mmol) was dissolved in 22.5 ml of 1,4-dioxane (CASNo.: 123-91-1) and added with 34 ml of 10% (w/w) aqueous Na₂CO₃ solutionin the form of suspension. 6.95 g of Fmoc-Cl (9-fluorenylmethylchloroformate, CAS No.: 28920-43-6, purchased from Energy Chemical, 26.8mmol) was dissolved in 34 ml of 1,4-dioxane, added into the abovesuspension in an ice bath, and naturally warmed to room temperature forreacting overnight. The reaction solution was poured into 150 ml of icewater, and extracted three times, each with 100 ml of methyl t-butylether, and the resultant organic phases were discarded. The aqueousphase remained was adjusted to pH 5 with concentrated hydrochloric acid,extracted twice, each with 100 ml of ethyl acetate. The obtained organicphases were combined and dried with anhydrous sodium sulfate. Thesolvent was removed by evaporation under reduced pressure to give 7.83 gof product PRO-7 as a white foamy solid. ¹H NMR (400 MHz, DMSO-d₆) δ7.91 (t, J=7.2 Hz, 2H), 7.67 (d, J=7.5 Hz, 2H), 7.48-7.39 (m, 2H),7.38-7.27 (m, 2H), 5.17 (s, 1H), 4.27 (s, 2H), 4.23-4.11 (m, 2H),3.55-3.41 (m, 3H), 2.31-2.10 (m, 1H), 2.08-1.88 (m, 1H). HRMS (ESI) m/zcalculated. for C₂₀H₁₉NO₅ [M−H]−352.1190, measured: 352.1033.

(12-1-1b) Synthesis of PRO-8

7.83 g of PRO-7 (22.2 mmol) was dissolved in 80 ml of THF (CAS No.:109-99-9), heated to 65° C. in an oil bath, added with 36.6 ml of 2mol/L solution of BH₃-Me₂S in THF (CAS No. 13292-87-0, purchased fromJ&K Scientific Ltd., 73.2 mmol) under reflux, and refluxed continuallyto react for 3 hours. The reaction solution was poured out, and theremaining solid was dissolved in methanol. To the resultant reactionsolution, methanol was added under stirring until no gas emits, stirredcontinually for 30 minutes. The solvent was removed by evaporation underreduced pressure, and then the residue was purified with petroleum etherthree times to give 7.1 g of product PRO-8 as a white solid. ¹H NMR (400MHz, DMSO-d6) δ 7.91 (t, J=6.7 Hz, 2H), 7.67 (d, J=7.2 Hz, 2H),7.49-7.39 (m, 2H), 7.38-7.26 (m, 2H), 5.18 (dd, J=6.1, 3.8 Hz, 1H), 4.28(s, 2H), 4.23-4.13 (m, 2H), 3.55-3.38 (m, 2H), 2.32-2.11 (m, 1H),2.08-1.89 (m, 1H). HRMS (ESI) m/z, calculated for C₂₀H₂₁NO₄ [M+Na]⁺362.1368, measured: 362.1012.

(12-1-1c) Synthesis of PRO-9

7.1 g of PRO-8 (21 mmol) was dissolved in 100 ml of pyridine, and addedwith 14.2 g of DMTr-Cl (4,4′-dimethoxytrityl chloride, 42 mmol) to reactunder stirring at room temperature for 5 hours. The solvent was removedby evaporation under reduced pressure. The resultant crude product wasdissolved in ethyl acetate and filtered to remove salt impurities. Thesolvent was removed by evaporation under reduced pressure, and then theresidue was purified by using a silica gel column. For purification, thecrude product dissolved in DCM was loaded onto the silica gel columnpretreated with pyridine to alkalify the column. DMTr-Cl was eluted withDCM containing 1% (v/v) pyridine, and then the product was eluted withethyl acetate. The eluate was collected, and the solvent was removed byevaporation under reduced pressure to give 8.2 g of product PRO-9 as awhite solid. HRMS (ESI) m/z, calculated for C₄₁H₃₉NO₆ [M+Na]+664.2675,measured: 664.2348; C18 RP-HPLC (Lot No.: JJS160324-1); purity: 94.20%.

(12-1-1d) Synthesis of PRO-10

8.2 g of PRO-9 (12.8 mmol) was dissolved in 64 ml of DMF and added with40 ml of piperidine (384 mmol) to react under stirring at roomtemperature for 30 minutes. The reaction solution was poured into 300 mlof ice water and extracted three times, each with 150 ml of ethylacetate. The resultant organic phases were combined and washed with 200ml of saturated brine, and the organic phase resulted from washing wasdried with anhydrous sodium sulfate. The solvent was removed byevaporation under reduced pressure, and then the residue was purified byusing a silica gel column. For purification, the crude product dissolvedin DCM was loaded onto the silica gel column pretreated with pyridine toalkalify the column. Fmoc was eluted with DCM containing 1% (v/v)pyridine, and then the product was eluted with ethyl acetate. The eluatewas collected, and the solvent was removed by evaporation under reducedpressure to give 4.65 g of product PRO-10 as a white solid. ¹H NMR (400MHz, DMSO-d₆) δ 7.40 (d, J=7.2 Hz, 2H), 7.35-7.18 (m, 7H), 6.93-6.84 (m,4H), 4.56 (d, J=3.9 Hz, 1H), 4.12 (s, 1H), 3.74 (s, 6H), 3.46-3.37 (m,1H), 2.88 (ddd, J=18.5, 10.0, 5.5 Hz, 2H), 2.75 (dd, J=8.7, 5.8 Hz, 1H),2.62 (dd, J=11.0, 2.7 Hz, 1H), 1.74-1.65 (m, 1H), 1.40 (ddd, J=12.9,8.5, 5.9 Hz, 1H); HRMS (ESI) m/z calculated for C₂₆H₂₉NO₄ [M+Na]⁺442.1994, measured: 442.1999; C18 RP-HPLC (Lot No.: JJS160329-1),purity: 97.07%.

(12-1-2) Synthesis of FIN-1

GAL-5 (4.5 g, 10 mmol) obtained according to the method described instep (2-1-1) was dissolved in 40 ml of DMF, sequentially added with 3.9g of DIEA (N,N-diisopropylethylamine, CAS No.: 7087-68-5, purchased fromAladdin Inc., 30 mmol) and 3.8 g of HBTU(benzotriazol-N,N,N′,N′-tetramethyluronium hexafluorophosphate, CAS No.:94790-37-2, purchased from Aladdin Inc., 11 mmol), and stirred at roomtemperature for 10 minutes. PRO-10 (4.2 g, 10 mmol) obtained in step(11-1-1d) was dissolved in 40 ml of DMF, and then added into the abovereaction solution. The resultant reaction solution was dried by additionof anhydrous sodium sulfate and stirred at room temperature for 2 hours.The reaction solution was poured into 120 ml of ice water and extractedthree times, each with 60 ml of ethyl acetate. The resultant organicphases were combined, washed with 20 ml of water and 20 ml of saturatedbrine, respectively. The organic phase obtained from washing wasisolated and dried with anhydrous sodium sulfate. The solvent wasremoved by evaporation under reduced pressure, and then the residue waspurified by using a silica gel column. For purification, a sample wasloaded onto the silica gel column pretreated with pyridine to alkalifythe column, and was eluted with dichloromethane (DCM) solutioncontaining 1% (v/v) triethylamine and 1% (v/v) methanol. The eluate wascollected, and the solvent was removed by evaporation under reducedpressure to give 6.5 g of product FIN-1 as a light yellow foamy solid.¹H NMR (400 MHz, DMSO-d₆) δ 7.83 (d, J=9.2 Hz, 1H), 7.32 (t, J=6.6 Hz,4H), 7.20 (td, J=8.9, 3.5 Hz, 5H), 6.93-6.84 (m, 4H), 5.21 (d, J=3.2 Hz,1H), 5.04-4.90 (m, 2H), 4.49 (s, 1H), 4.40 (d, J=4.4 Hz, 0.8H), 4.31 (d,J=5.0 Hz, 0.2H), 4.15 (s, 1H), 4.03 (s, 3H), 3.93 (s, 1H), 3.74 (s, 7H),3.59 (dt, J=12.0, 6.0 Hz, 1H), 3.50-3.40 (m, 1H), 3.39-3.25 (m, 3H),3.13 (dd, J=8.9, 5.2 Hz, 1H), 3.00 (dq, J=9.3, 5.3, 4.3 Hz, 1H), 2.22(s, 2H), 2.07 (s, 3H), 1.99 (s, 3H), 1.90 (s, 4H), 1.74 (s, 3H), 1.50(s, 3H), 1.36 (s, 1H). C18 RP-HPLC (Lot Number: LJ160422), purity:95.45%.

(12-1-3) Synthesis of FIN-2

FIN-1 (3.0 g, 3.53 mmol) obtained in step (12-1-2) and acetonitrile wereheated for azeotropic dehydration, subjected to suction drying underreduced pressure, dissolved in 10 ml of DMF (dried by immersing in amolecular sieve), added with 2.13 g of PA(bis(diisopropylamino)(2-cyanoethoxy)phosphine, Adamas Inc., product No.11356B, 7.06 mmol)) and 346 mg tetrazole (CAS No.: 288-94-8, purchasedfrom Aladdin Inc., 4.94 mmol) under nitrogen atmosphere, and stirred toreaction at room temperature. The reaction was supplemented with 10 mlof DMF and continually stirred to react for 1 hour. The solvent wasremoved by evaporation under reduced pressure, and then the residue waspurified by silica gel column chromatography. For purification, thecrude product dissolved in DCM was loaded onto the silica gel columnpretreated with pyridine to alkalify the column, and eluted with ethylacetate. The eluate was collected, and the solvent was removed byevaporation under reduced pressure to give 4.5 g of crude product as acolorless syrup. The crude product was completely dissolved in 50% (v/v)aqueous acetonitrile solution and purified by using a medium pressurecolumn (C-18, 330 g, 300 Å) pretreated with a solution of 1% (v/v)pyridine in acetonitrile to alkalify the column. A product peak wascollected by gradient elution and the solvent was removed by evaporationunder reduced pressure to give 2.2 g of product FIN-2 conjugatingmolecule as a white powder. ³¹P NMR (162 MHz, CDCl₃) δ 148.04, 147.94,147.62, 147.19, purity of ³¹P NMR: 92%; purity of C18 RP-HPLC: 90.54%.

(12-2) Linking FIN-2 Conjugating Molecule to a Solid Phase Support

The conjugation group (FIN_FIN_FIN) was linked to the 3′ terminal of thesense strand of RNA by linking the FIN-2 conjugating molecule obtainedin step (12-1-3) to a universal solid phase support (UnyLinker™ loadedNittoPhase® HL Solid Supports) by using the nucleic acid solid phasesynthesis method through three reaction cycles.

The linking of conjugation group FIN_FIN_FIN was proformed according tothe method described in Rajeev et al., Chem Bio Chem 2015, 16, 903-908.Specifically, the hydroxy protecting group was initially removed fromthe above-mentioned universal solid phase support and then the solidphase support, which was subsequently brought into contact and coupledwith the FIN-2 conjugating molecule under coupling reaction condition inthe presence of a coupling agent, and a FIN conjugating molecule linkedto the solid phase support was obtained after the capping and oxidationreaction. Moreover, the hydroxy protecting group DMTr was removed fromthe FIN conjugating molecule linked to the solid phase support, and thesolid phase support was further brought into contact and coupled withanother FIN-2 conjugating molecule, followed by capping and oxidationreaction. By repeating the above steps ofDeprotection-Coupling-Capping-Oxidation, a third FIN-2 conjugatingmolecule was linked, and thus a conjugation group (FIN_FIN_FIN) linkedto the solid phase support was obtained.

In the reactions described above, the reaction conditions of thedeprotection, coupling, capping and oxidation as well as the amounts ofthe solvents and agents are the same as those of the above solid phasesynthesis method of nucleic acid in Preparation Example 1.

(12-3) Synthesis of Conjugates F1-F5

The subject conjugates were prepared by the same methods as those insteps (2-2), (2-3A) and (2-4) of Preparation Example 2, except that: 1)the sense strand was synthesized starting from the compound obtained instep (12-2); and 2) the conjugated siRNAs had the sequencescorresponding to Conjugates A20-A23, B16-B17, C14, D14, E18-E20 and F20and Comparative Conjugates A1 and B1 as shown in Tables 4A-4G.

The molecular weight was measured by LC-MS instrument (LiquidChromatography-Mass Spectrometry, purchased from Waters Corp., Model:LCT Premier). The results showed that the measured values were inconformity with the calculated values, and thus it was confirmed thatthe synthesized conjugates were the designed compounds of interest,which have a structure as shown by Formula (307).

Preparation Example 13 Preparation of Comparative Conjugates A3, E2 andF2

In this Preparation Example, Comparative Conjugates A3, E2 and F2 weresynthesized. The conjugated siRNAs in these conjugates had sequencesshown in Tables 4A, 4E and 4F.

(13-1) Synthesis of (GalNAc)₃ Conjugating Molecule

Compound 30, i.e., the conjugating molecule containing the abovementioned linker -(L^(A))₃-trihydroxymethyl aminomethane-L^(B)- and thetargeting group N-acetylgalactosamine molecule (wherein each L^(A) canbe linked to one N-acetylgalactosamine molecule such that one linker canbe linked to three N-acetylgalactosamine molecules), was synthesizedaccording to the preparation method described in WO2014025805A1. Thisconjugating molecule can also be referred to as (GalNAc)₃ conjugatingmolecule, and the structure of compound 30 was shown as follows:

(13-2) Linking (GalNAc)₃ Conjugating Molecule to a Solid Phase Support

The (GalNAc)₃ conjugating molecule was linked to a solid phase supportby the same method as that in step (2-1-9) of Preparation Example 2,thereby obtaining (GalNAc)₃ conjugating molecule linked to a solid phasesupport.

(13-3) Synthesis of Comparative Conjugates A3, E2 and F2

Comparative Conjugates A3, E2 and F2 were prepared by the same methodsas those in steps (2-2), (2-3A) and (2-4) of Preparation Example 2,except that: 1) the sense strand was synthesized starting from thecompound obtained in step (13-2); and 2) the conjugated siRNAs hadsequences shown under NOs. A3, E2 and F2 in Tables 4A, 4E and 4F.

The molecular weight was measured by LC-MS instrument (LiquidChromatography-Mass Spectrometry, purchased from Waters Corp., Model:LCT Premier). The results showed that the measured values were inconformity with the calculated values, and thus it was confirmed thatthe synthesized conjugates were the designed compounds of interest,which have a structure as shown by Formula (305).

After the preparation of the above conjugates of the present disclosure,they were lyophilized to solid powder via standard process and storeduntil used. When being used, they can be reconstituted with, forexample, water for injection to a solution at a desired concentration.

The properties of the above siRNA and siRNA conjugates of the presentdisclosure prepared were studied by the examples below.

The effect experiments of the siRNA conjugates in Table 4A areillustrated as follows.

Experimental Example A1—the Toxicity of the siRNA Conjugates of thePresent Disclosure

In C57BL/6J mice, Conjugate A1 (0.9 wt % NaCl aqueous solution,administration volume of 10 mL/kg, concentrations of 10 mg/mL and 20mg/mL, wherein each concentration was used for 6 mice: three male andthree female) was subcutaneously administered to each mouse, with asingle dose of 100 mg/kg or 200 mg/kg (based on siRNA). Continuousclinical observation was performed during treatment period, which showsno animal death and no clinical symptoms associated with adverse drugresponses. 24 h after the administration, blood samples were taken forclinical pathology test and the mice were dissected. The results showthat no abnormalities were found in clinical pathology test and grossanatomy. Thus, the above results indicate the conjugates of the presentdisclosure have a relatively low toxicity at animal level.

Experimental Example A2 this Experiment Illustrated the Stability of thesiRNA Conjugates of the Present Disclosure (Experimental Example A2-1)Stability of the siRNA Conjugates of the Present Disclosure in theLysosome Lysate In Vitro

Preparation of test samples treated with the lysosome lysate:Comparative Conjugate A1 and Conjugate A21 (each provided in the form of0.9 wt % NaCl aqueous solution in which the concentration of siRNA is 20μM, 6 μl for each group) were individually mixed well with 27.2 μL ofsodium citrate aqueous solution (pH 5.0), 4.08 μL of deionized water and2.72 μL of Tritosomes (purchased from Xenotech Inc., Cat No. R0610LT,Lot No. 1610069), and incubated at a constant temperature of 37° C. 5 μLsamples were taken at each time point of 0 h, 1 h, 2 h, 4 h, 6 h, 8 h,24 h and 48 h respectively, added to 15 μL of 9 M urea for denaturation,and added with 4 μL of 6× loading buffer (purchased from Solarbio Inc.,Cat No. 20160830), then immediately cryopreserved in a −80° C. freezerto quench the reaction. 0 h represents the moment when the sample wastaken immediately after the samples to be tested are mixed well with thelysosome lysate.

Preparation of control samples untreated with the lysosome lysate: 1.5μL each of the conjugates above at equal moles (20 μM) was mixed wellwith 7.5 μL of sodium citrate aqueous solution (pH 5.0) and 1 μL ofdeionized water, added to 30 μL of 9 M urea solution for denaturation,and added with 8 μL of 6×loading buffer, then immediately cryopreservedin a −80° C. freezer to quench the reaction. The control sample for eachconjugate is marked as Con in the electrophoretogram.

16 wt % of non-denatured polyacrylamide gel was prepared. 20 μL each ofthe test samples and the control samples described above was loaded ontothe gel to perform electrophoresis under 20 mA constant current for 10minutes and then under 40 mA constant current for 30 minutes. Afterfinishing the electrophoresis, the gel was placed on a shaker andstained with Gelred dye (BioTium, Cat No. 13G1203) for 10 minutes. Thegel was subjected to imaging, observation and photocopying. The resultsare shown in FIG. 1 .

FIG. 1 shows the semiquantitative detection result of the in vitrostability of the tested siRNA conjugates in the Tritosome. The resultsindicate that the conjugates of the present disclosure can remainundegraded for a long time in Tritosome, showing good stability.

(Experimental Example A2-2) Stability of the siRNA Conjugates in theLysosome Lysate In Vitro

The stability was measured using the same method as that in ExperimentalExample 2-1, except that the samples to be tested are Conjugates A1, A6and Comparative siRNA1, and the time period of incubation withTritosomes is 0 h, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h and 8 h,respectively.

The results of the electrophoresis of non-denatured polyacrylamide gelare shown in FIG. 2 .

FIG. 2 shows the semiquantitative detection result of the in vitrostability of the tested siRNA conjugates in the Tritosome. The resultsindicate that the conjugates of the present disclosure can remainundegraded for a long time in Tritosome, showing good stability.

As can be seen from the results of FIGS. 1 and 2 , the siRNAs havingspecific modification according to the present disclosure exhibitsatisfactory stability in the lysosome lysate in vitro.

(Experimental Example A2-3) Stability in Human Plasma

Conjugates A1, A6, and Comparative siRNA2 (each provided in the form of0.9 wt % NaCl aqueous solution in which the concentration of siRNA is 20μM, 12 μl for each group) were individually mixed well with 108 μL of90% human plasma (diluted in PBS) and incubated at a constanttemperature of 37° C. 10 μL samples were taken at each time point of 0h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h and 72 h, respectively, andimmediately frozen in liquid nitrogen and cryopreserved in a −80° C.freezer. After sampling at each time point, each cryopreserved samplewas diluted 5-fold with 1×PBS (pH 7.4) and then taken in a volume of 10μL for use. Meanwhile, each of the samples to be tested was taken atequal moles (2 μM, 2 μL) and mixed well with 8 μL of 1×PBS (pH 7.4),thus obtaining 10 μL of samples untreated with human plasma (marked asCon). 20 wt % of non-denatured polyacrylamide gel was prepared. Eachcryopreserved sample above was mixed with 4 μL of loading buffer(aqueous solution of 20 mM EDTA, 36 wt % glycerol, and 0.06 wt %bromophenol blue) and then loaded onto the above gel to performelectrophoresis under 80 mA constant current for 60 minutes. Afterfinishing the electrophoresis, the gel was stained with 1×Sybr Gold dye(Invitrogen, Cat No. 11494) for 15 minutes followed by imaging. Theresults are shown in FIG. 3 .

FIG. 3 shows the semiquantitative detection result of the in vitrostability of the tested conjugates in human plasm.

As can be seen from the results of FIG. 3 , in human plasma, theconjugates of the present disclosure remain undegraded at up to 72hours, showing excellent stability in human plasma.

(Experimental Example A2-4) Stability of the Conjugates in the MonkeyPlasma

Conjugates A1, A6, and Comparative siRNA2 (each provided in the form of0.9 wt % NaCl aqueous solution in which the concentration of siRNA is 20μM, 12 μl for each group) were individually mixed well with 108 μL of90% cynomolgus monkey plasma (Monkey plasma, purchased form HONGQUANBio, Cat No. HQ70082, diluted in PBS) and incubated at a constanttemperature of 37° C. 10 μL samples were taken at each time point of 0h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h and 72 h, respectively, andimmediately frozen in liquid nitrogen and cryopreserved in a −80° C.freezer. After sampling at each time point, each sample was diluted5-fold with 1×PBS (pH 7.4) and then taken in a volume of 10 μL for use.Meanwhile, each of the samples to be tested was taken at equal moles (2μM, 2 μL) and mixed well with 8 μL of 1×PBS (pH 7.4), thus obtaining 10μL of samples untreated with monkey plasma (marked as Con). 20 wt % ofnon-denatured polyacrylamide gel was prepared. Each cryopreserved samplewas all mixed with 4 μL of loading buffer (aqueous solution of 20 mMEDTA, 36 wt % glycerol, and 0.06 wt % bromophenol blue) and then loadedonto the above gel to perform electrophoresis under 80 mA constantcurrent for 60 minutes. After finishing the electrophoresis, the gel wasstained with 1×Sybr Gold dye (Invitrogen, Cat No. 11494) for 15 minutesfollowed by imaging. The results are shown in FIG. 4 .

FIG. 4 shows the semiquantitative detection result of the in vitrostability of the tested siRNA in the monkey plasma.

As can be seen from the results of FIG. 4 , in cynomolgus monkey plasma,the siRNA conjugates of the present disclosure remain undegraded at upto 72 hours, showing excellent stability in monkey plasma.

(Experimental Example A2-5) this Experiment Illustrated the Stability ofthe siRNA Conjugates of the Present Disclosure in the Lysosome Lysate InVitro

1) Detection of the Stability in Murine Lysosome Lysate

Preparation of test samples treated with the lysosome lysate: ConjugateA2 and Comparative siRNA2 (20 μM, each 6 μL) were individually mixedwell with 27.2 μL of sodium citrate aqueous solution (pH 5.0), 4.08 μLof deionized water and 2.72 μL of murine lysosome lysate (Rat LiverTritosomes, purchased from Xenotech Inc., Cat No. R0610.LT, Lot No.1610069, at a final concentration of acid phosphatase of 0.2 mU/μL), andincubated at a constant temperature of 37° C. 5 μL mixed solution wastaken at each time point of 0 h, 1 h, 2 h, 4 h, 6 h, and 24 h,respectively, added to 15 μL of 9 M urea solution for denaturation, andadded with 4 μL of 6× loading buffer (purchased from Solarbio Inc., CatNo. 20160830), then immediately cryopreserved in a −80° C. freezer toquench the reaction. 0 h represents the moment when the sample was takenimmediately after the samples to be tested are mixed well with thelysosome lysate.

Preparation of control samples untreated with the lysosome lysate: 1.5μL each of the Conjugate A2 and Comparative siRNA2 (20 μM) at equalmoles was mixed well with 7.5 μL of sodium citrate aqueous solution (pH5.0) and 1 μL of deionized water, added to 30 μL of 9 M urea solutionfor denaturation, and added with 8 μL of 6×loading buffer, thenimmediately cryopreserved in a −80° C. freezer to quench the reaction.The control sample for each conjugate is marked as M to be compared withthe electrophoresis results of the sample.

16 wt % of non-denatured polyacrylamide gel was prepared. 20 μL each ofthe test sample and the control sample described above was loaded ontothe gel to perform electrophoresis under 20 mA constant current for 10minutes and then under 40 mA constant current for 30 minutes. Afterfinishing the electrophoresis, the gel was placed on a shaker andstained with Gelred dye (BioTium, Cat No. 13G1203) for 10 minutes. Thegel was subjected to imaging, observation and photocopying. The resultsare shown in FIG. 5 .

2) Detection of the Stability in Human Lysosome Lysate

The stability of Comparative siRNA2 and Conjugate A2 in the humanlysosome lysate was measured using the same method as that in 1), exceptthat the murine lysosome lysate was replaced with the human lysosomelysate (Human Liver Lysosomes, purchased from Xenotech Inc., Cat No.R0610.L, Lot No. 1610316). The results are shown in FIG. 6 .

The results indicate that the siRNA conjugates of the present disclosurecan remain undegraded for at least 24 hours both in human-originatedlysosome lysate and in murine lysosome lysate, showing satisfactorystability.

Experimental Example A3 the Results of the Pharmacokinetic Study ofConjugate A1 of the Present Disclosure in Rats In Vivo

In this Experimental Example, Conjugate A1 was administered to rats ineach experimental group (10 rats in each group, five male and fivefemale) by subcutaneous injection, respectively, with a single dose of10 mg/kg and 50 mg/kg. Subsequently, the drug concentration in plasma,liver and kidney tissues of rats were measured at each time point.

The SD rats used in this experimental example were provided by BeijingVital River Laboratory Animal Technology Co., Ltd.

Firstly, SD rats were randomly divided into groups according to the bodyweight and gender by using the PRISTIMAdata system version 7.2.0, andthen respectively administered with each group of the conjugatesaccording to the designed dosage. The drug dosages for all animals werecalculated according to the body weigh (single administration(subcutaneously), administration dosage of 10 mg/kg and 50 mg/kg, in theform of 0.9% NaCl aqueous solution containing 1 mg/ml and 5 mg/mlconjugates, and administration volume of 10 mL/kg). Rat whole blood wascollected from the jugular vein before administration and at 5 minutes(±30 seconds), 30 minutes (±1 minute), 1 hour (±2 minutes), 2 hours (±2minutes), 6 hours (±5 minutes), 24 hours (±10 minutes), 48 hours (±20minutes), 72 hours (±20 minutes), 120 hours (±30 minutes), and 168 hours(±30 minutes) after administration. Then the whole blood samples werecentrifugated at 1800×g at 2-8° C. for 10 minutes to separate plasma.About 70 μL volume of the plasma sample was placed in one tube, and theremaining of the sample was placed in another, both of which werecryopreserved at −70° C. to −86° C. for detection. Liver and kidneytissues of rats were collected at about 24, 48, 72, 120, and 168 hoursafter administration by the method comprising anesthetizing the ratswith pentobarbital sodium according to the weight thereof (60 mg/kg,intraperitoneal injection), euthanizing the rats by blood collectionfrom abdominal aorta, and performing gross anatomy. The liver and kidneyof each rat were sampled and stored in 1 mL cryotube at below −68° C.until detection and analysis.

The concentrations of the Conjugate A1 in plasma, liver and kidneytissues of rats were detected quantitatively by High Performance LiquidChromatography with Fluorescence Detection (HPLC-FLD) according to thefollowing steps:

(1) grinding the tissue until a tissue mass of no more than 80 mg wasobtained, then adding Tissue and Cell Lysis Solution (supplier:Epicentre, Cat No. MTC096H) to prepare a tissue homogenate of 66.7mg/mL;

(2) subjecting the tissue homogenate to a sonication (150 W, 30 s) todisrupt cells;

(3) for tissue samples, adding 75 μL of tissue samples to a 96-well PCRplate, adding 5 μL of proteinase K (supplier: Invitrogen, Cat No.25530-015) and 10 μL of mixed aqueous solution of 10 wt % acetonitrileand 0.01 wt % Tween 20; for plasma samples, adding 20 μL of plasma to a96-well PCR plate, adding 45 μL of Tissue and Cell Lysis Solution, 5 μLof proteinase K, and 20 μL of mixed aqueous solution of 10 wt %acetonitrile and 0.01 wt % Tween 20;

(4) blocking the plates and placing them in a PCR instrument (supplier:Applied Biosystems, model: GeneAmp® PCR system 9700) and incubating at65° C. for 45 minutes;

(5) after finishing incubation, adding 10 μl of 3 M KCl aqueous solution(supplier: Sigma-aldrich, Cat No. 60135-250ML), shaking well, andcentrifuging at 3200 rcf at 4° C. for 15 minutes;

(6) for tissue samples, adding 80 μL of supernatant into 120 μL ofhybridization mixture solution (formula: 0.5 mL of 6 μM PNA probe(supplier: TAHE-PNA), 1 mL of 200 mM Trizma/pH=8, 5 mL of 8 M ureaaqueous solution, 3.5 mL of H₂O, 2 mL of acetonitrile); for plasmasamples, adding 40 μL of supernatant into 160 μL of hybridizationmixture solution (formula: 0.5 mL of 6 μM PNA probe, 1 mL of 200 mMTrizma/pH=8, 5 mL of 8 M urea aqueous solution, 7.5 mL of H₂O, 2 mL ofacetonitrile);

(7) blocking the plates and placing them in a PCR instrument, incubatingat 95° C. for 15 minutes, then immediately placing on ice for 5 minutes;

(8) transferring the samples to new 96-well plates with conical bottom,shaking well, and centrifuging at 3200 rcf for 1 minute;

(9) injecting the samples for detection and quantitatively analyzing byusing HPLC-FLD (liquid-phase system supplier: Thermo Fisher,chromatography model: ultimate 3000).

The analyzed results can be found in FIGS. 7-10 , which show metaboliccurves over time of PK/TK plasma concentrations in rat plasma and PK/TKtissue concentrations in rat liver and kidney for Conjugate A1 at adosage of 10 mg/kg or 50 mg/kg, respectively. Specifically,

FIG. 7 is a metabolic curve over time showing PK/TK plasma concentrationfor Conjugate A1 in rat plasma at a dosage of 10 mg/kg.

FIG. 8 is a metabolic curve over time showing PK/TK tissueconcentrations for Conjugate A1 in rat liver and kidney at a dosage of10 mg/kg.

FIG. 9 is a metabolic curve over time showing PK/TK plasma concentrationfor Conjugate A1 in rat plasma at a dosage of 50 mg/kg.

FIG. 10 is a metabolic curve over time showing PK/TK tissueconcentrations for Conjugate A1 in rat liver and kidney at a dosage of50 mg/kg.

As can be seen from the results of FIGS. 7-10 , the concentrations forConjugate A1 in rat plasma were rapidly decreased below the detectionlimit within several hours, while the concentrations in rat liver tissuewere maintained at a relatively high and stable level over at least 168hours, either at a low dosage (10 mg/kg) or at a relatively high dosage(50 mg/kg). This shows that the siRNA conjugate of the presentdisclosure can be specifically and significantly enriched in liver andremain stable, showing a high degree of targeting.

Experimental Example A4—this Experiment Illustrated the InhibitoryEfficiency of the RNA Conjugates of the Present Disclosure Against theExpression of HBV mRNA In Vivo

In this experimental example, the inhibition efficiency of Conjugates A5and A7 against the expression of HBV mRNA in HBV transgenic miceC57BL/6J-Tg(Alb1HBV)44Bri/J was investigated.

HBsAg content in mouse serum was measured using Hepatitis B VirusSurface Antigen Assay Kit (Enzyme-linked Immunosorbent Assay, ELISA)(Shanghai Kehua Bio-engineering Co., Ltd.). Mice with S/COV>10 wereselected and randomly divided into groups (all female, 4 mice in eachgroup) and respectively numbered as Conjugate A5 and Conjugate A7, and anormal saline (NS) group was added as a control group. The drug dosagesfor all animals were calculated according to the body weight (singleadministration (subcutaneously), administration dosage of 1 mg/kg and0.1 mg/kg, in the form of 0.9% NaCl aqueous solution containing 0.2mg/ml and 0.02 mg/ml conjugates, and administration volume of 5 mL/kg).Animals were sacrificed on day 14 after administration. The liver wascollected and kept with RNA later (Sigma Aldrich), and the liver tissuewas homogenized with a tissue homogenizer. Then the total RNA wasextracted and obtained by using Trizol according to the standardprocedures for total RNA extraction.

The expression level of HBV mRNA in liver tissue was detected byreal-time fluorescent qPCR. Specifically, the extracted total RNA wasreverse transcribed into cDNA by using ImProm-II™ reverse transcriptionkit (Promega) according to the instruction, and then the inhibitoryefficiency of siRNAs against the expression of HBV mRNA in liver tissuewas detected by using the fluorescent qPCR kit (Beijing CowinBiosicences Co., Ltd). In this fluorescent qPCR method, β-actin gene wasused as an internal control gene, the HBV and β-actin were detected byusing primers for HBV and β-actin, respectively.

Sequences of primers for detection are shown in Table 5A.

TABLE 5A Sequences of primers for detection Upstream Downstream GenesPrimers Primers HBV 5′-CCGTCT 5′-TAATCTCC GTGCCTTCT TCCCCCAACTC CATCT-3′C-3′ (SEQ ID (SEQ ID NO: 425) NO: 426) β-actin 5′-AGCTTC 5′-TTCTGACCTTTGCAGCT CATTCCCACCA CCTTCGTT TCACA-3′ G-3′ (SEQ ID (SEQ ID NO: 428)NO: 427)

In this fluorescent qPCR method, the expression of HBV mRNA wasexpressed as the remaining expression of HBV X gene and calculated bythe following equation:

The remaining expression of HBV X gene=(the copy number of HBV X gene inthe test group/the copy number of β-actin gene in the test group)/(thecopy number of HBV gene in the control group/the copy number of β-actingene in the control group)×100%, which is marked as HBV X/β-actin mRNAexpression in the figures.

Then, the inhibition percentage of the conjugate against mRNA wascalculated according to the equation:

The inhibition percentage of the conjugate against mRNA=(1−the remainingexpression of HBV X gene)×100%,

wherein the control group was a group of control mice administered withNS in this experiment and each test group was a group of miceadministered with different siRNA conjugates, respectively. The resultsare shown in FIG. 11 .

In other experiments, several tests were further performed according tothe following conditions:

Tests were performed by using the same method described above, exceptthat the siRNA conjugate administered was replaced with Conjugates A1and A6 for testing, and the data were collected on day 14. The resultsare shown in FIG. 12 ; and

Tests were performed by using the same method described above, exceptthat the siRNA conjugate administered was replaced with Conjugates A1,A2, A3 and A4 for testing (5 mice in each group), and the data arecollected on day 28. Each conjugate was administered in the two dosagesof 1 mg/kg and 0.3 mg/kg (wherein the administration volume remained thesame, while the concentrations of the conjugate solutions wererespectively adjusted). The results are respectively shown in FIG. 13 .

Tests were performed by using the same method described above, exceptthat the siRNA conjugate administered was replaced with Conjugate A1 andComparative Conjugate A3 for testing, and the data are collected on day14. Each conjugate was administered in the two dosages of 1 mg/kg and0.1 mg/kg (wherein the administration volume remained the same, whilethe concentrations of the conjugate solutions were respectivelyadjusted). The results are respectively shown in FIG. 14 .

As can be seen from the above results, in several experiments withdifferent testing time points, all conjugates of the present disclosuredescribed above show high inhibitory activity against the expression ofHBV mRNA in mice in vivo.

Experimental Example A5 This experiment illustrated a test about therelationship between time and inhibitory efficiency of the siRNAconjugates of the present disclosure against the expression of HBsAg andHBV DNA in HBV transgenic mice serum.

An AAV-HBV mouse was used. After successful establishment of the animalmodels, these mice were randomly divided into groups based on HBsAgcontent in serum (5 mice in each group). Conjugate A1, ComparativeConjugate A2, Comparative Conjugate A3 and NS as a blank control wererespectively administered to each group. The drug dosages for allanimals were calculated according to the body weight (singleadministration (subcutaneously), administration dosage of 3 mg/kg and 1mg/kg, in the form of 0.9% NaCl aqueous solution containing 0.3 mg/mland 0.1 mg/ml conjugates, and administration volume of 5 mL/kg). Theblood was taken from mouse orbital venous plexus before administration(marked as DO) and on days 7, 14, 21, 28, 56, 84, 112, 140, 154, 168 and182 after administration, and HBsAg level in serum was measured for eachtime point. During the experiment, the detection of a subject is endedif the HBsAg content in serum in the test result is close to or morethan the original value.

About 100 μl orbital blood was taken each time, and the serum was noless than 20 μl after centrifugation. The expression level of HBsAg inserum was measured by using HBsAg CLIA kit (Autobio, CL0310). Theexpression level of HBV DNA was measured by extraction of the DNA fromthe serum with reference to the instruction of QIAamp 96 DNA Blood Kitfollowed by qPCR.

The normalized HBsAg expression level=(the content of HBsAg afteradministration/the content of HBsAg before administration)×100%.

The inhibition percentage against HBsAg=(1−the content of HBsAg afteradministration/the content of HBsAg before administration)×100%, whereinthe content of HBsAg was expressed in equivalents (UI) of HBsAg permilliliter (m1) of serum.

The normalized HBV DNA expression level=(the content of HBV DNA afteradministration/the content of HBV DNA before administration)×100%.

The inhibition percentage against HBV DNA=(1−the content of HBV DNAafter administration/the content of HBV DNA before administration)×100%,

wherein the content of HBV DNA was expressed in copies of HBV DNA permilliliter (m1) of serum.

The results are shown in FIGS. 15 and 16 .

As can be seen from the results of FIG. 15 , the NS negative controlgroup showed no inhibitory effect at different time points afteradministration; in contrast, each conjugate showed excellent inhibitoryeffect on HBsAg at different time points after administration. Inparticular, Conjugate A1 consistently showed high inhibition percentageagainst HBsAg in serum over a period of up to 140 days, indicatingstable and effective inhibition against the expression of HBV gene overa longer time period.

As can be seen from the results of FIG. 16 , the Conjugate A1 alsoshowed efficient inhibition against the expression of HBV DNA andmaintained higher inhibition percentage over a period of up to 84 days.

In contrast, although Comparative Conjugates A2 and A3 achieved similarmRNA inhibitory effects to the individual conjugates in the experimentsin vivo, the duration of the inhibitory effects as shown in FIGS. 15 and16 were significantly shorter than that of Conjugates 1 and 6 at thesame dose level.

According to the same methods as described above, two more tests werefurther performed, wherein serum HBsAg was measured, except that:

In M-Tg models, the administration doses of Conjugate A6 are 5 mg/kg, 1mg/kg and 0.2 mg/kg, and Comparative Conjugate A3 is 5 mg/kg; the testcontinued until day 78; and the results are shown in FIG. 17 ;

In 1.28 copy model mouse, the administration doses of Conjugate A1 are 3mg/kg and 1 mg/kg; the test continued until day 210; and the results areshown in FIGS. 18 and 19 .

For the various administration doses described above, each conjugate wasadministered in the same administration volume, while concentration ofthe solution was correspondingly adjusted, so as to be administered inthe corresponding dose.

From the results of FIGS. 15-19 , it can be seen that the siRNAconjugates of the present disclosure showed consistent and efficientinhibitory efficiency on serum HBsAg in various animal models, andregular dose dependency.

Experimental Example A6 This experiment illustrated that the siRNAconjugate of the present disclosure not only has higher activity invitro, but also shows low off-target effect (A6-1) HEK293A cells used inthis experimental example were provided by Nucleic Acid TechnologyLaboratory, Institute of Molecular Medicine, Peking University andcultured in DMEM complete media (Hyclone company) containing 20% fetalbovine serum (FBS, Hyclone company), 0.2 v % Penicillin-Streptomycin(Gibco, Invitrogen company) at 37° C. in an incubator containing 5%CO₂/95% air.

In this experimental example, Conjugate A1 was investigated in in vitropsiCHECK system for the on-target activity and off-target effect.Specifically, Conjugate A1 was tested for the activity of targetingcompletely matching target sequence (of which the nucleotide sequence iscompletely complementary with the full length nucleotide sequence of thesense/antisense strand of Conjugate A1) or targeting matching targetsequence in seed region (of which the nucleotide sequence iscomplementary with the nucleotide sequence of positions 1-8 of thesense/antisense strand of Conjugate A1).

According to the method described by Kumico Ui-Tei et. al., Functionaldissection of siRNA sequence by systematic DNA substitution: modifiedsiRNA with a DNA seed arm is a powerful tool for mammalian genesilencing with significantly reduced off-target effect. Nucleic AcidsResearch, 2008.36(7), 2136-2151, plasmids for detection were constructedand co-transfected with the siRNA conjugates to be detected into HEK293Acells; and the expression levels of the dual luciferase reporter genereflect the on-target activity and off-target effect of the siRNAconjugates. Specific steps are as follows:

[1] Construction of Plasmids for Detection

Four recombinant plasmids were constructed using psiCHECK™-2 (Promega™)plasmid, in which GSCM represents the on-target plasmid; and PSCM, GSSMand PSSM represent the off-target plasmids:

(1) GSCM, containing a target sequence, wherein the target sequence isfully complementary with all 21 nucleotide sequences of the antisensestrand in the conjugate to be detected (which is Conjugate A1 in thisexample).

(2) PSCM, containing a target sequence, wherein the target sequence isidentical with all 21 nucleotide sequences of the antisense strand inthe Conjugate A1.

(3) GSSM, containing a target sequence, wherein the target sequence isfully complementary with the nucleotide sequence at positions 1-8 from5′ terminal of the antisense strand in the Conjugate A1, while theremaining part of the target sequence corresponds to the nucleotidesequence at positions 9-21 from 5′ terminal of the antisense strand inthe Conjugate A1, but is completely mismatched; that is, when thenucleotide at any position in positions 9-21 from 5′ terminal of theantisense strand in the Conjugate A1 is G, C, A or U, the nucleotide atthe corresponding position in the target sequence is T, A, C or G.

(4) PSSM, containing a target sequence, wherein the target sequence isfully complementary with the nucleotide sequence at positions 1-8 fromthe 5′ terminal of the sense strand in the Conjugate A1, while theremaining part of the target sequence corresponds to the nucleotidesequence at positions 9-19 from 5′ terminal of the sense strand in theConjugate A1, but is completely mismatched; that is, when the nucleotideat any position in positions 9-19 from 5′ terminal of the sense strandin the Conjugate A1 is G, C, A or U, the nucleotide at the correspondingposition in the target sequence is T, A, C or G. In order to have thesame length as the target sequence in GSSM, two CC were added at 3′terminal of the target sequence in PSSM.

The target sequence was inserted into the Xho I/Not I site of thepsiCHECK™-2 plasmid.

[2] Transfection

In a 96-well plate, siRNA and each of the above plasmids wereco-transfected according to the instruction of Lipofectamine™ 2000(Invitrogen), each plasmid corresponding to several specificconcentrations of Conjugate A1. Specifically, 10 ng of plasmid wastransfected per well, using 0.2 μL of Lipofectamine™ 2000 per well; forthe on-target plasmid GSCM, the final concentration (based on theconcentration of siRNA) of Conjugate A1 was from 100 nM to 0.0001 nM(4-fold serial dilutions of 11 concentrations), 3 replicate wells pergroup.

[3] Detection

24 hours after co-transfection, the HEK293A cells were lysed by using adual luciferase reporter gene assay kit (Promega, Cat No. E2940)according to the instruction to detect the expression level of the dualluciferase reporter gene. For the test group of each specificconcentration, those untreated with the conjugate were used as control(con). The Renilla luciferase protein level (Ren) was normalized to thefirefly luciferase protein level (Fir).

The dose-response curves were plotted by the activity results measuredat different siRNA concentrations, and the curves were fitted using thefunction log(inhibitor) vs. response-Variable slope of Graphpad 5.0software. The IC₅₀ of the siRNA targeting GSCM was calculated based onthe dose-response curve with the formula below:

$Y = {{Bot} + \frac{{Top} - {Bot}}{1 + \text{?}}}$?indicates text missing or illegible when filed

wherein:

Y is the expression level of remaining mRNA,

X is the logarithm of the concentration of transfected siRNA,

Bot is the Y value at the bottom of the steady stage,

Top is the Y value at the top of the steady stage,

Log IC₅₀ is the X value at which Y is the median value between thebottom and the top of the steady stage, and HillSlope is the slope ofthe curve.

The IC₅₀ of the Conjugate A1 targeting GSCM was determined viacalculation based on the dose-response curve. The results are shown inFIGS. 20A-20D, which indicate that the IC₅₀ value of Conjugate A1corresponding to GSCM was 0.0019 nM. Conjugate A1 corresponding to PSCM,GSSM or PSSM shows no significant inhibitory effect at each siRNAconcentration, indicating that the siRNA conjugate of the presentdisclosure not only has higher activity in vitro, but also exhibits lowoff-target effect.

According to the above results, Conjugate A1 shows superior inhibitoryeffect on the expression of the target mRNA in the on-target plasmidwith low IC₅₀; while shows no inhibitory effect on the expression of thethree off-target plasmids. Thus, Conjugate A1 not only has superiorinhibitory efficiency of the target mRNA, but also exhibits lowoff-target effect.

The effect experiment of the siRNA conjugates in Table 4B wasillustrated as follows.

Experimental Example B1 this Experiment Illustrated Inhibitory ActivityIn Vitro of the siRNA Conjugates of the Present Disclosure ExperimentalExample B1-1 On-Target Activity in In Vitro psiCHECK System

HEK293A cells used in this experimental example were provided by NucleicAcid Technology Laboratory, Institute of Molecular Medicine, PekingUniversity and cultured in DMEM complete media (Hyclone company)containing 20% fetal bovine serum (FBS, Hyclone company), 0.2 v %Penicillin-Streptomycin (Gibco, Invitrogen company) at 37° C. in anincubator containing 5% CO₂/95% air.

In this experimental example, Conjugates B16 and B17 were investigatedin in vitro psiCHECK system for the on-target activity and off-targeteffect. Specifically, Conjugates B16 and B17 were tested for theactivity of targeting completely matching target sequence (of which thenucleotide sequence is completely complementary with the full lengthnucleotide sequence of the sense/antisense strand of the conjugates) ortargeting matching target sequence in seed region (of which thenucleotide sequence is complementary with the nucleotide sequence ofpositions 1-8 of the sense/antisense strand of the conjugates).

According to the method described by Kumico Ui-Tei et. al., Functionaldissection of siRNA sequence by systematic DNA substitution: modifiedsiRNA with a DNA seed arm is a powerful tool for mammalian genesilencing with significantly reduced off-target effect. Nucleic AcidsResearch, 2008.36(7), 2136-2151, plasmids for detection were constructedand co-transfected with the siRNA conjugates to be detected into HEK293Acells; and the expression levels of the dual luciferase reporter genereflect the on-target activity of the siRNA conjugates. Specific stepsare as follows:

[1] Construction of a Plasmid for Detection

An on-target plasmid was constructed using psiCHECK™-2 (Promega™)plasmid. This plasmid contains a target sequence, which is fullycomplementarily paired with all 21 nucleotide sequences of the antisensestrand in the conjugate to be detected (i.e., Conjugate B16 or B17). Thetarget sequence was inserted into the Xho I/Not I site of thepsiCHECK™-2 plasmid.

[2] Transfection

In a 96-well plate, siRNA conjugate and the above plasmid wereco-transfected according to the instruction of Lipofectamine™ 2000(Invitrogen), respectively. Specifically, 10 ng of plasmid wastransfected per well, using 0.2 μL of Lipofectamine™ 2000 per well; thefinal concentrations (based on the concentration of siRNA) of theconjugates were 0.1 nM, 0.05 nM and 0.01 nM, and those untreated withthe conjugates in each group were used as control, with 3 replicatewells per group.

NC is a universal negative control B01001 with no homology to the targetgene sequence (GenePharma Co., Ltd).

[3] Detection

24 hours after co-transfection, the HEK293A cells were lysed by using adual luciferase reporter gene assay kit (Promega, Cat No. E2940)according to the instruction to detect the expression level of the dualluciferase reporter gene. The Renilla luciferase protein level (Ren) wasnormalized to the firefly luciferase protein level (Fir). The resultsare shown in FIG. 21 .

The results indicated that Conjugates B16 and B17 both have goodinhibitory activity in intro.

Experimental Example B1-2 On-Target Activity and Off-Target Effect in InVitro psiCHECK System

In this experimental example, on-target activity and off-target effectof Conjugate B2 in in vitro psiCHECK system was investigated.

Conjugate B2 was tested by the method described in Experimental ExampleA6, except that 4 target sequences were constructed which correspondingsto Conjugate B2; and the concentrations were diluted from 0.1 nM to0.0001 nM; each group of plasmids corresponds to 11 concentrations ofsiRNA. The test results are shown in FIG. 22 .

As can be seen from FIG. 22 , Conjugate B2 not only has excellentinhibitory effect on the target mRNA, but also exhibits low off-targeteffect.

Experimental Example B2 this Experiment Illustrated the Stability of thesiRNA Conjugates of the Present Disclosure (Experimental Example B2-1)Stability of the siRNA Conjugates in the Lysosome Lysate In Vitro

Preparation of test samples treated with the lysosome lysate: ConjugatesB1 and B2 (each provided in the form of 0.9 wt % NaCl aqueous solutionin which the concentration of siRNA is 20 μM, 6 μl for each group,respectively) were individually mixed well with 27.2 μL of sodiumcitrate aqueous solution (pH 5.0), 4.08 μL of deionized water and 2.72μL of Tritosomes (commercially available from Xenotech Inc., Cat No.R0610LT, Lot No. 1610069), and incubated at a constant temperature of37° C. 5 μL samples were taken at each time point of 0 h, 5 min, 15 min,30 min, 1 h, 2 h, 4 h, and 8 h respectively, added to 15 μL of 9 M ureaaqueous solution for denaturation, and added with 4 μL of 6× loadingbuffer (purchased from Solarbio Inc., Cat No. 20160830), thenimmediately cryopreserved in a −80° C. freezer to quench the reaction. 0h represents the moment when the sample was taken immediately after thesamples to be tested are mixed well with the lysosome lysate.

Preparation of control samples untreated with the lysosome lysate: 1.5μL for each of the corresponding conjugates (20 μM) at equal moles wasmixed well with 7.5 μL of sodium citrate aqueous solution (pH 5.0) and 1μL of deionized water, added to 30 μL of 9 M urea solution fordenaturation, and added with 8 μL of 6×loading buffer, then immediatelycryopreserved in a −80° C. freezer to quench the reaction. The controlsample is marked as Con in the electrophoretogram.

16 wt % of non-denatured polyacrylamide gel was prepared. 20 μL each ofthe test sample and the control sample described above was loaded ontothe gel to perform electrophoresis under 20 mA constant current for 10minutes and then under 40 mA constant current for 30 minutes. Afterfinishing the electrophoresis, the gel was placed on a shaker andstained with Gelred dye (BioTium, Cat No. 13G1203) for 10 minutes. Thegel was subjected to imaging, observation and photocopying. The resultsare shown in FIG. 23 .

FIG. 23 shows the semiquantitative detection result of the in vitrostability of the tested siRNA conjugates in the Tritosome. The resultsindicate that the conjugates of the present disclosure can remainundegraded for a long time in Tritosome, showing good stability.

(Experimental Example B2-2) Stability of the siRNA Conjugates in HumanPlasma

Conjugates B1 and B2 (each provided in the form of 0.9 wt % NaCl aqueoussolution in which the concentration of siRNA is 20 μM, 12 μl for eachgroup) were individually mixed well with 108 μL of 90% human plasma(diluted in PBS) and incubated at a constant temperature of 37° C. 10 μLsamples were taken at each time point of 0 h, 2 h, 4 h, 6 h, 8 h, 24 h,48 h and 72 h, respectively, and immediately frozen in liquid nitrogenand cryopreserved in a −80° C. freezer. After sampling at each timepoint, each cryopreserved sample was diluted 5-fold with 1×PBS (pH 7.4)and then taken in a volume of 10 μL for use. Meanwhile, the siRNAconjugate was taken at equal moles (siRNA concentration of 2 μM, 2 μL)and mixed well with 8 μL of 1×PBS (pH 7.4), thus obtaining 10 μL ofsample untreated with human plasma (marked as Con).

20 wt % of non-denatured polyacrylamide gel was prepared. Eachcryopreserved sample above was all mixed with 4 μL of loading buffer(aqueous solution of 20 mM EDTA, 36 wt % glycerol, and 0.06 wt %bromophenol blue) and then loaded onto the gel to performelectrophoresis under 80 mA constant current for 60 minutes. Afterfinishing the electrophoresis, the gel was stained with 1×Sybr Gold dye(Invitrogen, Cat No. 11494) for 15 minutes followed by imaging. Theresults are shown in FIG. 24 .

FIG. 24 shows the semiquantitative detection result of the in vitrostability of the tested conjugates in human plasm.

As can be seen from the results of FIG. 24 , the conjugates of thepresent disclosure remain undegraded at up to 72 hours in human plasma,showing excellent stability in human plasma.

(Experimental Example B2-3) Stability of siRNA Conjugates in the MonkeyPlasma

In another experiment, the stability of Conjugates B1 and B2 in monkeyplasma (Monkey plasma, purchased form HONGQUAN Bio, Cat No. HQ70082,diluted in PBS) was measured using the same method as that inExperimental Example 2-2. The results are shown in FIG. 25 .

FIG. 25 shows the semiquantitative detection result of the in vitrostability of the tested siRNA in the monkey plasma.

As can be seen from the results of FIG. 25 , in cynomolgus monkeyplasma, the siRNA conjugates of the present disclosure remain undegradedat up to 72 hours, showing excellent stability in monkey plasma.

Experimental Example B3 this Experiment Illustrated the Inhibition ofthe Conjugates of the Present Disclosure Against the Expression of HBVmRNA in Mice

In this experimental example, the inhibitory efficiency of Conjugate B1against the expression level of HBV mRNA in HBV transgenic miceC57BL/6J-Tg (Alb1HBV) 44Bri/J were investigated.

At first, C57BL/6J-Tg (Alb1HBV) 44Bri/J mice were randomly divided intogroups based on HBsAg content in serum (all female, 4 mice in eachgroup) and respectively numbered, and a normal saline (NS) group wasadded as a control group. The drug dosages for all animals werecalculated according to the body weight (single administration(subcutaneously), administration dosage of 1 mg/kg and 0.1 mg/kgConjugate B1, in the form of 0.9% NaCl aqueous solution containing 0.2mg/ml and 0.02 mg/ml conjugates, and administration volume of 5 mL/kg).Animals were sacrificed on day 7 after administration. The liver wascollected and kept with RNA later (Sigma Aldrich), and the liver tissuewas homogenized with a tissue homogenizer. Then the total RNA wasextracted and obtained by using Trizol according to the standardprocedure for total RNA extraction.

The expression level of HBV mRNA in liver tissue was measured byreal-time fluorescent qPCR. Specifically, the extracted total RNA wasreverse transcribed into cDNA by using ImProm-II™ reverse transcriptionkit (Promega) according to the instruction, and then the inhibitoryefficiency of siRNAs against the expression of HBV mRNA in liver tissuewas measured by using the fluorescent qPCR kit (Beijing CowinBiosicences Co., Ltd). In this fluorescent qPCR method, a gene encodingglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internalcontrol gene, the HBV and GAPDH were detected by using primers for HBVand GAPDH, respectively.

Sequences of primers for detection are shown in Table 5B.

TABLE 5B Sequences of primers for detection Upstream Downstream GenesPrimers Primers HBV 5′-CCGTCT 5′-TAATCTCC GTGCCTTCT TCCCCCAACTC CATCT-3′C-3′ (SEQ ID (SEQ ID NO: 425) NO: 426) GAPDH 5′-AACTTTG 5′-TGGAAGAGCATTGTGGA GTGGGAGTTG AGGGCTC-3′ CTGTTGA-3′ (SEQ ID (SEQ ID NO: 431)NO: 432)

In this fluorescent qPCR method, the inhibitory activity of siRNA wasexpressed as the remaining expression of HBV gene and calculated by thefollowing equation:

The remaining expression of HBV gene=(the copy number of HBV gene in thetest group/the copy number of GAPDH in the test group)/(the copy numberof HBV gene in the control group/the copy number of GAPDH in the controlgroup)×100%.

Then, the inhibition percentage against mRNA was calculated according tothe following equation:

The inhibition percentage against mRNA=(1−the remaining expression ofHBV gene)×100%,

wherein the control group was a group of control mice administered withNS in this experiment and each test group was a group of miceadministered with different siRNA conjugates, respectively. The resultsare shown in FIG. 26 .

In other experiments, two tests were further performed according to thefollowing conditions:

Tests were performed by using the same method described above, exceptthat the siRNA conjugate administered was replaced with Conjugate B2 fortesting, and the data were collected on day 7. The results are shown inFIG. 27 ; and

the siRNA conjugate administered was replaced with Conjugates B1 and B2for testing; the Conjugate B1 was administered respectively in the twodosages of 1 mg/kg and 0.1 mg/kg;

Conjugate B2 was administered in the dosage of 1 mg/kg; and thesequences for detection were replaced with the sequences shown in Table5C. The results are shown in FIG. 28 .

TABLE 5C  Sequences of primers for detection Upstream Downstream GenesPrimers Primers HBV S 5′-CGTTTCT 5′-CAGCGGT CCTGGCTCAG AAAAAGGGACTTTA-3′ TCAA-3′ (SEQ ID (SEQ ID NO: 429) NO: 430) GAPDH 5′-AACTTTGG5′-TGGAAGAGT CATTGTGGAAG GGGAGTTGCTG GGCTC-3′ TTGA-3′ (SEQ ID (SEQ IDNO: 431) NO: 432)

As can be seen from FIGS. 27 and 28 , all conjugates of the presentdisclosure described above show good inhibitory effect on the targetmRNA. Moreover, the inhibitory effects thereof against different kindsof HBV mRNA remain substantially the same.

Experimental Example B4 this Experiment Illustrated a Test about theRelationship Between Time and Inhibitory Efficiency of the siRNAConjugates of the Present Disclosure Against the Expression of HBsAg andHBV DNA in HBV Transgenic Mice Serum

For low-concentration AAV-HBV model mouse, the mice were randomlydivided into groups based on HBsAg content in serum (5 mice in eachgroup). Conjugate B2 and NS as a blank control were respectivelyadministered to each group. The drug dosages for all animals werecalculated according to the body weight (single administration(subcutaneously), administration dosage of 3 mg/kg and 1 mg/kg, in theform of 0.9% NaCl aqueous solution containing 0.6 mg/ml and 0.2 mg/mlconjugates, and administration volume of 5 mL/kg). The blood was takenfrom mouse orbital venous plexus before administration and on days 7,14, 21, 28, 56, 84, 98, 112, 126, and 140 after administration, andHBsAg level in serum was measured for each time point.

About 100 μl orbital blood was taken each time, and the serum was noless than 20 μl after centrifugation. The expression level of HBsAg inserum was measured by using HBsAg CLIA kit (Autobio, CL0310). Theexpression level of HBV DNA was measured by extraction of the DNA fromthe serum with reference to the instruction of QIAamp 96 DNA Blood Kitfollowed by qPCR.

The inhibition percentage against HBsAg was calculated according to thefollowing equation:

The inhibition percentage against HBsAg=(1−the content of HBsAg afteradministration/the content of HBsAg before administration)×100%, whereinthe content of HBsAg was expressed in equivalents (UI) of HBsAg permilliliter (m1) of serum.

The inhibition percentage against HBV DNA was calculated according tothe following equation:

The inhibition percentage against HBV DNA=(1−the content of HBV DNAafter administration/the content of HBV DNA before administration)×100%,

wherein the content of HBV DNA was expressed in copies of HBV DNA permilliliter (m1) of serum.

The results are shown in FIG. 29 .

As can be seen from the results of FIG. 29 , the NS negative controlgroup shows no inhibitory effect at different time points afteradministration; in contrast, Conjugate B2 shows excellent inhibitoryeffect on HBsAg at different time points after administration. Inparticular, Conjugate B2 consistently shows high inhibition percentageagainst HBsAg in serum over a period of up to 100 days, indicatingstable and effective inhibition against the expression of HBV gene overa longer time period.

According to the same methods as described above, further tests wereperformed. In 1.28 copy model mouse, the administration doses ofConjugate B2 are 3 mg/kg and 1 mg/kg, using 0.9 wt % NaCl aqueoussolution containing 0.6 mg/ml and 0.2 mg/ml conjugates, withadministration volume of 5 mL/kg. The administration period continueduntil day 85; and the inhibitory effects against HBsAg and HBV DNA weremeasured according to the method described above. The results are shownin FIGS. 30 and 31 .

As can be seen from FIGS. 30 and 31 , in 1.28 copy model mouse,Conjugate B2 of the present disclosure consistently showed highinhibition against the expression of HBV gene and HBV DNA over a periodof 85 days.

Hereinbelow, an experiment for verifying the properties of the siRNAconjugates shown in Table 4C is described:

Experimental Example C1 this Experiment Illustrated the Stability of thesiRNA Conjugates Shown in Table 4C (Experimental Example C1-1) Stabilityof the siRNA Conjugates in the Lysosome Lysate In Vitro

Preparation of test samples treated with the lysosome lysate: ConjugateC2 (provided in the form of 0.9 wt % NaCl aqueous solution in which theconcentration of siRNA is 20 μM, 6 μl for each group) was individuallymixed well with 27.2 μL of sodium citrate aqueous solution (pH 5.0),4.08 μL of deionized water and 2.72 μL of Tritosomes (purchased fromXenotech Inc., Cat No. R0610LT, Lot No. 1610069), and incubated at aconstant temperature of 37° C. 5 μL samples were taken at each timepoint of 0 h, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 hrespectively, added to 15 μL of 9 M urea aqueous solution fordenaturation, and added with 4 μL of 6× loading buffer (purchased fromSolarbio Inc., Cat No. 20160830) was added, then immediatelycryopreserved in a −80° C. freezer to quench the reaction. 0 hrepresents the moment when the sample was taken immediately after thesamples to be tested are mixed well with the lysosome lysate.

Preparation of control samples untreated with the lysosome lysate: 1.5μL of Conjugate C2 at equal moles (20 μM) was mixed well with 7.5 μL ofsodium citrate aqueous solution (pH 5.0) and 1 μL of deionized water,added to 30 μL of 9 M urea solution for denaturation, and added with 8μL of 6×loading buffer, then immediately cryopreserved in a −80° C.freezer to quench the reaction. The control sample is marked as Con inthe electrophoretogram.

16 wt % of non-denatured polyacrylamide gel was prepared. 20 μL each ofthe test sample and the control sample described above was loaded ontothe gel to perform electrophoresis under 20 mA constant current for 10minutes and then under 40 mA constant current for 30 minutes. Afterfinishing the electrophoresis, the gel was placed on a shaker andstained with Gelred dye (BioTium, Cat No. 13G1203) for 10 minutes. Thegel was subjected to imaging, observation and photocopying. The resultsare shown in FIG. 32 .

FIG. 32 shows the semiquantitative detection result of the in vitrostability of the tested siRNA conjugates in the Tritosome. The resultshows that the conjugate of the present disclosure can remain undegradedfor a long time in lysosome, showing good stability.

(Experimental Example C1-2) Stability of siRNA Conjugates in HumanPlasma

Conjugate C2 (provided in the form of 0.9 wt % NaCl aqueous solution at20 μM with regard to siRNA, 12 μl for each group) was individually mixedwell with 108 μL of 90% human plasma (diluted in PBS) and incubated at aconstant temperature of 37° C. 10 μL samples were taken at each timepoint of 0 h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h and 72 h, respectively, andimmediately frozen in liquid nitrogen and cryopreserved in a −80° C.freezer. After sampling at each time point, each cryopreserved samplewas diluted 5-fold with 1×PBS (pH 7.4) and then taken in a volume of 10μL for use. Meanwhile, the siRNA conjugate was taken at equal moles(siRNA concentration of 2 μM, 2 μL) and mixed well with 8 μL of 1×PBS(pH 7.4), thus obtaining 10 μL of samples untreated with human plasma(marked as Con).

20 wt % of non-denatured polyacrylamide gel was prepared. Eachcryopreserved sample above was all mixed with 4 μL of loading buffer(aqueous solution of 20 mM EDTA, 36 wt % glycerol, and 0.06 wt %bromophenol blue) and then loaded onto the gel to performelectrophoresis under 80 mA constant current for 60 minutes. Afterfinishing the electrophoresis, the gel was stained with 1×Sybr Gold dye(Invitrogen, Cat No. 11494) for 15 minutes followed by imaging. Theresults are shown in FIG. 33 .

As can be seen from the results of FIG. 33 , in human plasma, theconjugates of the present disclosure remained undegraded at up to 72hours, showing excellent stability in human plasma.

(Experimental Example C1-3) Stability of siRNA Conjugates in the MonkeyPlasma

In another experiment, the stability of Conjugate C2 in monkey plasma(Monkey plasma, purchased form HONGQUAN Bio, Cat No. HQ70082, diluted inPBS) was measured using the same method as that in Experimental ExampleC1-2. The results are shown in FIG. 34 .

FIG. 34 shows the semiquantitative detection result of the in vitrostability of the tested siRNA in the monkey plasma.

As can be seen from the results of FIG. 34 , in cynomolgus monkeyplasma, the siRNA conjugates of the present disclosure remainedundegraded at up to 72 hours, showing excellent stability in monkeyplasma.

Experimental Example C2 this Experimental Example Illustrated InhibitoryActivity In Vitro of the siRNA Conjugates of the Present Disclosure(Experimental Example C2-1) On-Target Activity in In Vitro psiCHECKSystem

Conjugate C14 was tested by the method described in Experimental ExampleB1-2, except that the target sequences were constructed using thesequences of Conjugate C14. The test results are shown in FIG. 35 . Theresults indicate that Conjugate C14 has good inhibitory activity invitro.

(Experimental Example C2-2) Measurement of IC₅₀ in In Vitro psiCHECKSystem

Conjugate C2 was tested by the method described in Experimental ExampleA6, except that the target sequences were constructed using thesequences of Conjugate C2; and the concentrations were diluted from 0.1nM to 0.0001 nM, each group of sequences was tested at 11concentrations. The results are shown in FIG. 36 .

As can be seen from the FIG. 36 , Conjugate C2 not only had excellentinhibitory effect on the target mRNA, but also exhibited low off-targeteffect.

Experimental Example C3—this Experimental Example Illustrated theInhibition of the Conjugates of the Present Disclosure AgainstExpression of HBV mRNA in Mice

In this experimental example, the inhibition efficiency of Conjugate C2against the expression level of HBV mRNA in HBV transgenic miceC57BL/6J-Tg(Alb1HBV)44Bri/J was investigated.

At first, C57BL/6J-Tg (Alb1HBV) 44Bri/J mice were randomly divided intogroups (all female, 4 mice in each group) and numbered individually, anda normal saline (NS) group was added as a control group. The drugdosages for all animals were calculated according to the body weight(single administration (subcutaneously), administration dosage of 1mg/kg and 0.1 mg/kg Conjugate C2, in the form of 0.9% NaCl aqueoussolution containing 0.2 mg/ml and 0.02 mg/ml conjugates, andadministration volume of 5 mL/kg). Animals were sacrificed on day 7after administration. The liver was collected and kept with RNA later(Sigma Aldrich), and the liver tissue was homogenized with a tissuehomogenizer. Then the total RNA was extracted and obtained by usingTrizol according to the standard procedure for total RNA extraction.

The expression level of HBV mRNA in liver tissue was measured byreal-time fluorescent qPCR. Specifically, the extracted total RNA wasreverse transcribed into cDNA by using ImProm-II™ reverse transcriptionkit (Promega) according to the instruction, and then the inhibitoryefficiency of siRNAs against the expression of HBV mRNA in liver tissuewas measured by using the fluorescent qPCR kit (Beijing CowinBiosicences Co., Ltd). In this fluorescent qPCR method, β-actin gene wasused as an internal control gene, the HBV and 0-actin were detected byusing primers for HBV and β-actin, respectively.

Sequences of primers for detection are shown in Table 5A.

In this fluorescent qPCR method, the inhibitory activity of siRNA wasexpressed as the remaining expression of HBV gene and calculated by thefollowing equation:

The remaining expression of HBV gene=(the copy number of HBV gene in thetest group/the copy number of β-actin gene in the test group)/(the copynumber of HBV gene in the control group/the copy number of β-actin genein the control group)×100%, which is marked as the expression of HBVX/β-actin mRNA in the figure.

Then, the inhibition percentage against mRNA was calculated according tothe following equation:

The inhibition percentage against mRNA=(1−the remaining expression ofHBV gene)×100%,

wherein the control group was a group of control mice administered withNS in this experiment and each test group was a group of miceadministered with different siRNA conjugates, respectively. The resultsare shown in FIG. 37 .

As can be seen from the results of FIG. 37 , the inhibition percentageof the conjugates of the present disclosure against the target mRNA wasup to 93.8%, exhibiting good inhibitory effect.

Experimental Example C4 this Experimental Example Illustrated theInhibitory Effect of the siRNA Conjugates of the Present Disclosure(Single Administration) Against HBsAg and HBV X mRNA in M-Tg Model Mice

HBV transgenic (M-TgHBV) mice (purchased from Department of Animal,Shanghai Public Health Center) were randomly divided into groups basedon HBsAg content in serum (6 mice in each group, all male) andrespectively numbered as a normal saline (NS) control group, ConjugateC2 1 mg/kg and 3 mg/kg groups. The drug dosages for all animals werecalculated according to the body weight (single administration(subcutaneously), and administration volume of 10 mL/kg). The blood wastaken from mouse orbital venous plexus before administration (marked asDO) and on days 7, 14, 21, 28, 42, 56, 70, and 85 after administration,and HBsAg level in serum was measured for each time point.

About 0.5 ml orbital blood was taken each time, and the serum was noless than 200 μl after centrifugation. The content of HBsAg in serum wasmeasured by using HBsAg CLIA kit (Autobio, CL0310).

The normalized HBsAg level in serum=(the content of HBsAg afteradministration in the test group/the content of HBsAg beforeadministration in the test group)×100%.

The inhibition percentage against HBsAg=(1−the content of HBsAg afteradministration/the content of HBsAg before administration)×100%, whereinthe content of HBsAg was expressed in equivalents (UI) of HBsAg permilliliter (m1) of serum.

The following FIGS. 38 and 39 show the test results of the inhibitoryeffect of the tested siRNA conjugate in a single administration againstHBsAg and HBV X mRNA in M-Tg model mice.

As can be seen from the results of FIGS. 38 and 39 , Conjugate C2administered at 3 mg/kg maintained higher inhibition percentage againstHBsAg over a period of 21 days, exhibiting an inhibition percentage ofup to 90% or higher; and Conjugate C2 administered at 3 mg/kg stillshowed higher inhibition percentage against HBV X mRNA on day 85.

Hereinbelow, an effect experiment of the siRNA conjugates shown in Table4D was described.

Experimental Example D1 this Experiment Illustrated Stability of thesiRNA Conjugates of the Present Disclosure Experimental Example (D1-1)Stability of the siRNA Conjugates in the Lysosome Lysate In Vitro

According to the method described in Experimental Example C1-1, thestability of Conjugate D2 in the lysosome lysate in vitro was measured.The results are shown in FIG. 40 .

FIG. 40 shows the semiquantitative detection result of the in vitrostability of the tested siRNA conjugates in the lysosome. The resultshowed that the conjugates of the present disclosure can remainundegraded for a long time in lysosome, showing good stability.

Experimental Example (D1-2) Stability of the siRNA Conjugates in theHuman/Monkey Plasma

According to the method described in Experimental Examples C₁-2 andC₁-3, the stability of Conjugate D2 respectively in the human plasma invitro and in the cynomolgus monkey plasma in vitro was measured. Theresults are shown in FIGS. 41 and 42 .

As can be seen from the results of FIGS. 41 and 42 , in the human/monkeyplasma, the conjugates of the present disclosure remained undegraded atup to 72 hours, showing excellent stability.

Experimental Example D2 this Experimental Example Illustrated InhibitoryActivity In Vitro of the siRNA Conjugates of the Present Disclosure(Experimental Example D2-1) On-Target Activity in In Vitro psiCHECKSystem

HEK293A cells as used herein were provided by Nucleic acid technologylaboratory, Institute of Molecular Medicine, Peking University andcultured in DMEM complete media (Hyclone company) containing 20% fetalbovine serum (FBS, Hyclone company), 0.2 v % Penicillin-Streptomycin(Gibco, Invitrogen company) at 37° C. in an incubator containing 5%CO₂/95% air.

Conjugate D14 was tested by the method described in Experimental ExampleA6, except that target sequences were constructed based on the sequencesof Conjugate D14; and for each sequence, the concentration was dilutedfrom 0.1 nM to 0.0001 nM, and each sequence was tested at 11concentrations. The results are shown in FIG. 43 , indicating thatConjugate D14 has good inhibitory activity in vitro.

(Experimental Example D2-2) Measurement of IC₅₀ in In Vitro psiCHECKSystem

In this experimental example, the IC₅₀ of Conjugate D2 in in vitropsiCHECK system was investigated.

Conjugate D2 was tested according to the method described inExperimental Example B1-2, except that four target sequences wereconstructed based on the sequences of Conjugate D2; and for eachsequence, the concentration was diluted from 0.1 nM to 0.0001 nM, andeach sequence was tested at 11 concentrations. The results are shown inFIG. 44 , indicating that Conjugate D2 not only has excellent inhibitoryeffect on the target mRNA, but also shows low off-target effects.

Experimental Example D3 this Experimental Example Illustrated theInhibition of the Conjugate of the Present Disclosure Against theExpression of HBV mRNA in Mice

In this experimental example, the inhibition efficiency of Conjugate D2against the expression level of HBV mRNA in HBV transgenic miceC57BL/6J-Tg(Alb1HBV)44Bri/J was investigated.

Conjugate D2 was tested using the method described in ExperimentalExample C3. The results are shown in FIG. 45 .

As can be seen from the results of FIG. 45 , the conjugates of thepresent disclosure showed good inhibitory effect on the target mRNA.Specifically, Conjugate D2 showed, at the dosage of 1 mg/kg, aninhibition percentage against the target mRNA of up to 93.63%; and atthe dosage of a much lower concentration (0.1 mg/kg), also resulted inan inhibition percentage of 77.05%, showing good inhibitory effect.

Experimental Example D4 this Experimental Example Illustrated theInhibitory Effect of the siRNA Conjugates of the Present DisclosureAdministered in a Single Dose Against HBsAg and HBV X mRNA in M-Tg ModelMice

HBV transgenic (M-TgHBV) mice (purchased from Department of Animal,Shanghai Public Health Center) were randomly divided into groups basedon HBsAg content in serum (6 mice in each group, all male) andrespectively numbered as a normal saline (NS) control group, ConjugateD2 1 mg/kg and 3 mg/kg groups. The drug dosages for all animals werecalculated according to the body weight (single administration(subcutaneously), and administration volume of 10 mL/kg). The blood wastaken from mouse orbital venous plexus before administration and on days7, 14, 21, 28, 42, 56, 70, and 85 after administration, and HBsAg levelin serum was measured for each time point.

About 0.5 ml orbital blood was taken each time, and the serum was noless than 200 μl after centrifugation. The content of HBsAg in serum wasmeasured by using HBsAg CLIA kit (Autobio, CL0310). The remaining amountof HBsAg expression was calculated according to the following equation:

The remaining amount of HBsAg expression=(the content of HBsAg in thetest group/the content of HBsAg in the NS control group)×100%, whereinthe content of HBsAg was expressed in equivalents (UI) of HBsAg permilliliter (ml) of serum.

The following FIGS. 46 and 47 show the test results of the inhibitoryeffect of the tested siRNA conjugate administered in a singleadministration against HBsAg and HBV X mRNA in M-Tg model mice.

As can be seen from the results of FIGS. 46 and 47 : Conjugate D2administered at 3 mg/kg maintained higher inhibition against HBsAg overa period of 50 days, exhibiting an inhibition percentage of 90% orhigher and the maximum inhibition percentage of 95% or higher; andConjugate D2 administered at 3 mg/kg still showed an inhibitionpercentage of 62% against HBV X mRNA on day 85.

Hereinbelow, an experiment for verifying the effects of the siRNAconjugates shown in Table 4E was described.

Experimental Example E1 Detection for the Inhibitory Activity andOff-Target Effect of siRNA in In Vitro psiCHECK System

In this experimental example, siRNAs E1, E4 and Comparative siRNA3 wereinvestigated in in vitro psiCHECK system for the on-target activity andoff-target effect. Specifically, the activities of three siRNA targetingcompletely matching target sequences or targeting matching targetsequence in seed region were tested, respectively.

Tests were conducted by using the method described in ExperimentalExample B1-2, except that four target sequences were constructed basedon the sequences to be tested; the testing concentration was dilutedfrom 5 nM to 0.00008 nM (3-fold serial dilutions), totaling 11concentrations. The inhibitory effects of Comparative siRNA3 against theexpressions of four recombinant plasmids were shown in FIGS. 48A-48D,while the inhibitory effects of siRNA E1 against the expressions of fourrecombinant plasmids were shown in FIGS. 49A-49D. As can be seen fromthese Figures, the unmodified Comparative siRNA3 at 5 nM exhibited aninhibition percentage of about 20% against the expressions of GSSM andPSCM, indicating low off-target effects both in the seed region of theantisense strand and in the sense strand. However, the modified siRNA E1provided by the present disclosure showed no off-target effect; andsiRNAE4, consistent with siRNA E1, showed no off-target effect.

The dose-response curves were plotted by the activity results measuredat different siRNA concentrations, and the curves were fitted using thefunction log(inhibitor) vs. response-Variable slope of Graphpad 5.0software. The IC₅₀ of the siRNA t targeting GSCM o be detected wascalculated below based on the dose-response curve with the formulabelow. The results are shown in Table 5:

$Y = {{Bot} + \frac{{Top} - {Bot}}{1 + \text{?}}}$?indicates text missing or illegible when filed

wherein

Y is the expression level of remaining mRNA,

X is the logarithm of the concentration of transfected siRNA,

Bot is the Y value at the bottom of the steady stage,

Top is the Y value at the top of the steady stage,

Log IC₅₀ is the X value at which Y is the median value between thebottom and the top of the steady stage, and HillSlope is the slope ofthe curve.

TABLE 6E IC₅₀ value of siRNA against GSCM IC₅₀ value against siRNA No.GSCM siRNA E1 siAN1M3SVP 0.017 nM siRNA E4 siAN1M3S 0.024 nM Comp. siRNA3 siAN1 0.0028 nM 

As can be seen from Table 6E, the modified siRNA of the presentdisclosure showed very high inhibitory activity in in vitro psiCHECKsystem, with IC₅₀ ranging between 3 and 30 μM. Meanwhile, even at aconcentration of 5 nM, no off-target effect was observed in the siRNAsto be detected.

Experimental Example E2 Detection of Inhibitory Activity of siRNA andsiRNA Conjugate in In Vitro Cell System

Experimental Example E2-1 Detection of the inhibitory efficiency ofsiRNA in Huh 7 cell against the expression level of ANGPTL 3 mRNA

The siRNAs (siRNA E1, E2 and E4 and Comparative siRNA4) to be detectedwere transfected to Human hepatoma cell lines Huh7 by usingLipofectamine™ 2000. The final concentrations of siRNAs were 5 nM, 0.25nM and 0.05 nM, respectively, 2 replicate wells per concentration. Cellsuntreated with siRNA were used as a blank control.

The expression levels of ANGPTL3 mRNAs in Huh 7 cells transfected withsiRNAs at various concentrations were measured by PCR (QuantitativeReal-Time PCR), respectively. Specific steps were as follows: 24 hoursafter cultivation of transfected cells, the total RNA was extracted andobtained by using Trizol (Thermo Fisher) according to the standardprocedure for total RNA extraction; 1 μg of the total RNA wasindividually extracted and reverse transcribed into cDNA by usingreverse transcription kit (Promega, Cat No. A3500) according to theinstruction thereof. The expression level of ANGPTL3 mRNA was detectedbased on the template cDNA according to the steps described in theinstruction by using 2×Ultra SYBR Mixture (with ROX) (Beijing CowinBiosicences Co., Ltd, Cat No. CW 0956). Therein, the PCR primers ofGAPDH for amplifying ANGPTL3 and as an internal control gene are shownin Table 5E.

TABLE 5E Primer Information Nucleotide SEQ Primer Sequences ID Genestypes (5′ →3′) NO Human Upstream ACCAACTATACGCTACAT 433 ANGPTL3 PrimerDownstream CCTCCTGAATAACCCTCT 434 Primer Human UpstreamGGTCGGAGTCAACGGATTT 435 GAPDH Primer Downstream CCAGCATCGCCCCACTTGA 436Primer

The expression level of ANGPTL3 mRNA was calculated by the followingequation:

the expression level of ANGPTL3 mRNA=(the expression level of ANGPTL3mRNA in the test group/the expression level of GAPDH mRNA in the testgroup)/(the expression level of ANGPTL3 mRNA in the control group/theexpression level of GAPDH mRNA in the control group)×100%.

The inhibition percentage of siRNA against the expression level ofANGPTL3 mRNA is (1−the expression level of ANGPTL3 mRNA)×100%. Therein,Huh7 cells individually treated with siRNAs at various concentrationswere used in the test groups, and Huh 7 cells untreated with siRNAs wereused in the control group (marked as “Blank” in FIG. 50A). The resultsare shown in FIG. 50A.

As can be seen from FIG. 50A, the modified siRNAs provided by thepresent disclosure showed higher inhibitory activity in the Huh7 celllines.

Experimental Example E2-2 Detection of the Inhibitory Efficiency ofsiRNA Conjugate Against the Expression Level of ANGPTL3 mRNA in Huh7Cells

Detection was conducted by the same method as in Experimental ExampleE2-1, except that the samples to be detected were Conjugates E18 andE19, and the final concentrations of the conjugates (calculated based onthe amount of siRNA) were 50 nM and 5 nM. The in vitro inhibitoryactivity of each conjugate is shown in FIG. 50B.

As can be seen from FIG. 50B, the siRNA conjugates provided by thepresent invention showed higher inhibitory activity in Huh7 cell lines,and the conjugates at 5 nM showed an inhibition percentage of 60-80%against the expression level of ANGPTL3 mRNA.

Experimental Example E2-3 Measurement of IC₅₀ of siRNA Conjugate AgainstANGPTL3 mRNA in Huh7 Cells

Detection was conducted by the same method as that in ExperimentalExample E2-1, except that the samples to be detected were Conjugates E18and E19. The final concentrations of the conjugates (calculated based onthe amount of siRNAs) were diluted five-fold from 50 nM to 0.016 nM,with the lowest concentration being set at 0.00001 nM (totaling 7concentrations), 3 replicate wells per group.

In further experiments, the sample to be detected was Conjugate E2. Thefinal concentration of the conjugate (calculated based on the amount ofsiRNA) was double diluted from 2 nM to 0.0078 nM (totaling 9concentrations), 2 replicate wells per group.

In still further experiments, the samples to be detected were conjugatesE1 and E4. The final concentrations of the conjugates (calculated basedon the amount of siRNA) were double diluted from 0.5 nM to 0.03125 nM,with the highest concentration being set at 5 nM (totaling 6concentrations), 2 replicate wells per group.

The IC₅₀ value was calculated by the same method as that in ExperimentalExample 1 according to the measured inhibition percentages of siRNAconjugates at different concentrations against the expression level ofANGPTL3 mRNA. The IC₅₀ value of the conjugates to be detected in invitro Huh7 cells can be obtained. The results are shown in Table 7E.

TABLE 7E IC₅₀ of siRNA conjugates against ANGPTL3 mRNA Conjugate NO.IC₅₀ Conjugate E18 FIN-siAN1M3SVP 0.0851 nM Conjugate E19 FIN-siAN2M3SVP0.1419 nM Conjugate E2 L10-siAN1M3SP 0.1271 nM Conjugate E1L10-siAN1M3SVP 0.2137 nM Conjugate E4 L10-siAN1M3S 0.3833 nM

As can be seen from Table 7E, the siRNA conjugates of the presentinvention showed very high inhibitory activity in in vitro cell lines,with IC₅₀ ranging from 0.085 to 0.383 nM.

Experimental Example E3 Detection of the Stability of siRNAs and siRNAConjugates in Plasma and Lysosome Experimental Example E3-1 Detection ofthe Stability of siRNAs in Lysosome

In this experimental example, the stability of siRNAs E1, E2 and E4 inmurine lysosome lysate were investigated.

Preparation of test samples treated with the lysosome lysate: 6 μL eachof the siRNAs (at 20 μM) was individually mixed well with 27.2 μL ofsodium citrate aqueous solution (pH 5.0), 4.08 μL of deionized water and2.72 μL of murine lysosome lysate (Rat Liver Tritosomes, purchased fromXenotech Inc., Cat No. R0610LT, Lot No. 1610069, at a finalconcentration of acid phosphatase of 0.2 mU/μL), and incubated at aconstant temperature of 37° C. 5 μL samples were taken at each timepoint of 0 h, 1 h, 2 h, 4 h, 6 h, and 24 h respectively, added to 15 μLof 9 M urea aqueous solution for denaturation, and added with 4 μL of 6×loading buffer (purchased from Solarbio Inc., Cat No. 20160830), thenimmediately cryopreserved in a −80° C. freezer to quench the reaction. 0h represents the moment when the sample was taken immediately after thesamples to be tested are mixed well with the lysosome lysate.

Preparation of control samples untreated with the lysosome lysate: 1.5μL each of the conjugates at equal moles (20 μM) was mixed well with 7.5μL of sodium citrate aqueous solution (pH 5.0) and 1 μL of deionizedwater, added to 30 μL of 9 M urea solution for denaturation, and addedwith 8 μL of 6×loading buffer, then immediately cryopreserved in a −80°C. freezer to quench the reaction. The control sample for each siRNA wasmarked as Con in the electrophoretogram.

16 wt % of non-denatured polyacrylamide gel was prepared. 20 μL each ofthe test samples and the control samples described above was loaded ontothe gel to perform electrophoresis under 20 mA constant current for 10minutes and then under 40 mA constant current for 30 minutes. Afterfinishing the electrophoresis, the gel was placed on a shaker andstained with Gelred dye (BioTium, Cat No. 13G1203) for 10 minutes. Thegel was subjected to imaging, observation and photocopying. The resultsare shown in FIG. 51A.

As can be seen from FIG. 51A, the modified siRNAs of the presentinvention remained stable for at least 24 hours in murine lysosome.

Experimental Example E3-2 Detection of the Stability of siRNA Conjugatesin Lysosome

In this experimental example, the stability of Conjugates E1 and E4 inmurine lysosome lysate was investigated.

Detection was conducted according to the same method as that inexperimental example 3-1, except that: the samples to be detected wereConjugates E1 and E4, and the concentration of the conjugates werecalculated based on the amount of siRNA, and the time points fordetection were 0 h, 5 minutes, 15 minutes, 30 minutes, 1 h, 2 h, 4 h and6 h. The gel image was shown in FIG. 51B.

As can be seen from FIG. 51B, the siRNA conjugates of the presentinvention remained undegraded for at least 1 hour in murine lysosome,and then the major band of the electrophoresis was only slightly shifteddownward. In view of the high stability of the corresponding siRNA inthe lysosome lysate, it is considered that the downward shifting of theband may be the cleavage of a monosaccharide on the conjugation group.The siRNA conjugates of the present disclosure show satisfactorystability.

Experimental Example E3-3 Detection of the Stability of siRNA Conjugatesin Plasma

In this experimental example, the stability of Conjugates E1 and E4 inhuman plasma was investigated.

Conjugates E1 and E4 and Comparative siRNA3 (the concentration of siRNAor siRNA conjugate is 20 μM, 12 μl, and the conjugate is calculatedbased on the amount of siRNA) were individually mixed well with 108 μLof 90% human plasma (diluted in PBS) and incubated at a constanttemperature of 37° C. 10 μL samples were taken at each time point of 0h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h and 72 h, respectively, andimmediately frozen in liquid nitrogen and cryopreserved in a −80° C.freezer for use. After sampling at each time point, each cryopreservedsample was diluted 5-fold with 1×PBS (pH 7.4) and then taken in a volumeof 10 μL for use. Meanwhile, the siRNAs (2 μM, 2 μL) or siRNA conjugate(at the siRNA concentration of 2 μM, 2 μL) was taken at equal moles andmixed well with 8 μL of 1×PBS, thus obtaining 10 μL of samples untreatedwith human plasma (marked as M).

20 wt % of non-denatured polyacrylamide gel was prepared. Each of theabove samples was mixed with 4 μL of loading buffer (aqueous solution of20 mM EDTA, 36 wt % glycerol, and 0.06 wt % bromophenol blue) and thenloaded onto the gel to perform electrophoresis for under 80 mA constantcurrent about 60 minutes. After finishing the electrophoresis, the gelwas placed on the shaker and stained with 1×Sybr Gold dye (Invitrogen,Cat No. 11494) for 15 minutes followed. The gel was subjected toimaging, observation and photocopying. The results are shown in FIG.51C.

As can be seen from FIG. 51C, the siRNA conjugates of the presentinvention remained undegraded at up to 72 hours in human plasma, showingexcellent stability in human plasma.

In further experiments, the stability of Conjugates E1 and E4 in monkeyplasma was detected by using the same method described above. Theresults are shown in FIG. 51D.

The results indicate that the siRNA conjugates of the present inventioncan stay stably for at least 72 hours both in human plasma and in monkeyplasma, showing excellent stability.

Experimental Example E4 Detection of the Inhibitory Efficiency of siRNAConjugates Against the Expression Level of ANGPTL3 mRNA in Mice In Vivo,and Detection of the Inhibitory Effect on Blood Lipid ExperimentalExample E4-1 Determination of ED₅₀ of siRNA Conjugates Against ANGPTL3mRNA in Normal Mice C57 In Vivo

In this experimental example, the inhibitory activity of Conjugates E18and E19 in normal mice C57 in vivo was investigated.

Normal mice C57 of 6-8 weeks old were randomly divided into groups (5mice in each group). Conjugates E18, E19 and PBS were individuallyadministered to the mice in each group. The drug dosages for all animalswere calculated according to the body weight (single administration(subcutaneously), administration dosage of 10 mg/kg, 3 mg/kg, 1 mg/kg,0.3 mg/kg and 0.1 mg/kg for each siRNA conjugate (calculated based onthe amount of siRNA)). Moreover, the lowest dosage for Conjugates E18and E19 was 0.003 mg/kg. Each test group was administered in theadministration volume of 10 mL/kg. Each siRNA conjugate was individuallyadministered in the form of PBS aqueous solution. The drug concentrationof the conjugate to be formulated was calculated according toadministered dosage and volume. Mice were sacrificed on day 3 afteradministration. The liver was collected and kept with RNA later (SigmaAldrich), and the liver tissue was homogenized with a tissuehomogenizer. Then the total RNA was extracted and obtained by usingTrizol (Thermo Fisher) according to the standard procedure for total RNAextraction.

The expression level of ANGPTL3 mRNA in liver tissue was measured byreal-time fluorescent qPCR. Specifically, cDNA was obtained by reversetranscription using reverse transcription kit (Promega, Cat No. A3500)according to the instruction thereof. The expression level of ANGPTL3mRNA was measured based on the template cDNA according to the steps inthe instruction by using 2×Ultra SYBR Mixture (with ROX) (Beijing CowinBiosicences Co., Ltd, Cat No. CW 0956). Therein, the PCR primers ofGAPDH for amplifying ANGPTL3 and as an internal control gene are shownin Table 8E.

TABLE 8E sequences of primers Nucleotide Sequences Genes SEQ ID NO. (5′ →3′) Mouse 437 GAGGAGCAGCTAACCAACTTAAT ANGPTL3 438TCTGCATGTGCTGTTGACTTAAT Mouse 439 AACTTTGGCATTGTGGAAGGGCTC GAPDH 440TGGAAGAGTGGGAGTTGCTGTTGA

The expression level of ANGPTL3 mRNA was calculated by the equation: theexpression level of ANGPTL3 mRNA=[(the expression level of ANGPTL3 mRNAin the test group/the expression level of GAPDH mRNA in the testgroup)/(the expression level of ANGPTL3 mRNA in the control group/theexpression level of GAPDH mRNA in the control group)]×100%.

The inhibition percentage of the conjugates against the expression levelof ANGPTL3 mRNA was calculated by the equation:

the inhibition percentage=[1−(the expression level of ANGPTL3 mRNA inthe test group/the expression level of β-actin mRNA in the testgroup)/(the expression level of ANGPTL3 mRNA in the control group/theexpression level of β-actin mRNA in the control group)×100%.

Therein, the control group was a group of control mice administered withPBS in this experiment and each test group was a group of miceadministered with different siRNA conjugate, respectively.

ED₅₀ was calculated by the same method as in Experimental Example E1according to the inhibition percentages of siRNA conjugates at differentconcentrations against the expression level of ANGPTL3 mRNA. The ED₅₀value of the conjugates to be detected in normal mice in vivo can beobtained. The results are shown in Table 9E.

TABLE 9E ED₅₀ of siRNA conjugates against the ANGPTL3 mRNA in livertissue of normal mice c57 Conjugates NO. ED50 ConjugateE18FIN-siAN1M3SVP 0.1403 nM ConjugateE19 FIN-siAN2M3SVP 0.1595 nM

As can be seen from Table 9E, the inhibitory activity of the testedconjugates in normal mice in vivo was highly consistent with that of thecorresponding conjugates in in vitro cell line described in ExperimentalExample 2-3, with ED50 ranging between 0.1403 and 0.1595 nM, indicatingthat the siRNA conjugates of the present invention showed very highinhibitory activity in normal mice in vivo.

Experimental Example E4-2 Inhibitory Efficiency of the siRNA ConjugatesAgainst the Expression Level of ANGPTL3 mRNA in Normal Mice BALB/c InVivo, and the Effect on Blood Lipid

In this experimental example, the inhibitory percentage of ConjugatesE18 and E20 against ANGPTL3 mRNA in liver tissue in normal mice BALB/cin vivo, and the effect on blood lipid were investigated.

Normal mice BALB/c of 6-8 weeks old were randomly divided into groups(10 mice in each group). Conjugates E18, E20, Comparative Conjugate E2,and PBS were individually administered to the mice in each group. Thedrug dosages for all animals were calculated according to the bodyweight (single administration (subcutaneously), two administrationdosage of 3 mg/kg and 0.3 mg/kg for the siRNA conjugates (calculatedbased on the amount of siRNA), and the administration volume of 10mL/kg). Each siRNA conjugate was administered in the form of PBS aqueoussolution. The drug concentration of the conjugate to be formulated wascalculated according to administered dosage and volume. The blood wastaken from mouse orbital vein before administration and on days 7 and 14after administration, the blood lipid level in serum was tested at eachtime point. Five mice were respectively sacrificed on days 7 and 14after administration, and the liver tissue was collected to detect theexpression level of ANGPTL3 mRNA in liver.

About 100 μl orbital blood was taken each time, and the serum wasobtained after centrifugation. The contents of total cholesterol (CHO)and triglyceride (TG) in serum were further measured by using aPM1P000/3 full-automatic serum biochemical analyzer (SABA, Italy).

The normalized blood lipid level=(the blood lipid content in the testgroup after administration/the blood lipid content in the test groupbefore administration)×100%.

Inhibition percentage against blood lipid level=(1−the blood lipidcontent in the test group after administration/the blood lipid contentin the test group before administration)×100%.

Blood lipid refers to total cholesterol or triglyceride.

The blood lipid contents of mice on day 7 after administration are shownin FIGS. 52A-52B, and the blood lipid contents of mice on day 14 afteradministration are shown in FIGS. 52C-52D.

As can be seen from FIGS. 52A-52D, the tested siRNA conjugatessignificantly reduced the blood lipid level in normal mice. On day 14after administration, the siRNA conjugates of the present invention atthe dose of 3 mg/kg showed stronger ability to reduce blood lipid levelas compared with the positive control (Comparative Conjugate E2).

The inhibitory efficiency of siRNA conjugates against the expressionlevel of ANGPTL3 mRNA in liver was measured by real-time fluorescentqPCR using the same method as that in Experimental Example E4-1. Theresults are shown in Table 10E.

TABLE 10E inhibitory efficiency of siRNA conjugates against ANGPTL3 mRNAin liver tissue of normal mice BALB/c Inhibition Inhibition percentagepercentage against against Dose mRNA on mRNA on Conjugates NO. (mg/kg)day 7 (%) day 14 (%) Comparative (GalNAc)₃-65695 3 96.6 91.2 ConjugateE2 Conjugate E18 FIN-siAN1M3SVP 3 96.7 97.4 Conjugate E20 FIN-siAN1M3S 398.3 95.8 Conjugate E18 FIN-siAN1M3SVP 0.3 70.6 46.5 Conjugate E20FIN-siAN1M3S 0.3 68.0 34.0

In further experiments, the blood lipid and the expression level ofANGPTL3 mRNA were measured by the same methods described above, exceptthat the administered conjugates were Conjugates E1, E4 and ComparativeConjugate E2; and the time for detection was on days 14 and 28 afteradministration. The inhibitory effect of each conjugate against ANGPTL3mRNA is shown in FIGS. 53A-53D, and the inhibitory effect on blood lipidis shown in FIGS. 54A-54D.

As can be seen from FIGS. 53A-53D, on day 14 after administration, thesiRNA conjugates of the present disclosure at high dose showed aninhibition percentage of up to 95% against ANGPTL3 mRNA, i.e. showinginhibitory intensity significantly higher than Comparative Conjugate E2.For the siRNA conjugates at low doses, the tested siRNA conjugates,observed for an extended time period until day 28 after administration,all showed strong inhibitory effect on ANGPTL3 mRNA in liver tissue ofnormal mice, and the inhibitory intensity was significantly higher thanthat of the Comp. Conjugate.

As can be seen from FIGS. 54A-54D, in the serum of the mice treated withsiRNA conjugates of the present invention, the contents of both CHO andTG were reduced significantly, and blood lipid level reduction was atleast observed until 28 days after administration. The siRNA conjugatesof the present disclosure at a dose of 3 mg/kg showed stronger abilityto reduce blood lipid level than the positive control (ComparativeConjugate E2).

Experimental Example E4-3 Inhibitory Efficiency of siRNA ConjugatesAgainst the Expression Level of ANGPTL3 mRNA in Obese Mice In Vivo andthe Effect on Blood Lipid

In this experimental example, the inhibition percentage of Conjugate E18against ANGPTL3 mRNA in liver tissue of ob/ob mice in vivo and theeffect on blood lipid were investigated.

The expression level of ANGPTL3 mRNA and the blood lipid of ob/ob micewere measured by the same method described in Experimental Example E4-2,except that: ob/ob mice of 6-8 weeks old (6 mice in each group) wereadopted; the conjugate administered was Conjugate E18, of which theadministration dosages were 3 mg/kg, 1 mg/kg and 0.3 mg/kg; and theblood was taken two days before administration (marked as day −2) and onday 7, 14, 21, 28 and 34 after administration; the mice were sacrificedon day 34. The inhibitory effects of Conjugate E18 on blood lipid areshown in FIGS. 55A-55B; and the inhibitory effect on ANGPTL3 mRNA isshown in FIG. 55C.

As can be from the figures, in the mice treated with siRNA conjugates ofthe present invention, the contents of both CHO and TG were reducedsignificantly, and certain blood lipid reduction effect was at leastobserved until 34 days after administration. Meanwhile, on day 34 afteradministration, the siRNA conjugate can still effectively inhibit theexpression of ANGPTL3 mRNA.

Experimental Example E4-4 Effect of siRNA Conjugates on Blood Lipid inHigh-Fat Model Mice In Vivo

In this experimental example, the inhibition percentage of Conjugate E1against ANGPTL3 mRNA in liver tissue of human APOC3 transgenic mice andthe effect thereof on blood lipid were investigated.

Human APOC3 transgenic mice Tg(APOC3)3707Bre were randomly divided intogroups based on TG content>2 mmol/L in serum (6 mice in each group).Conjugate E1, Comparative Conjugate E1 and PBS blank control wereindividually administered to the mice in each group. The drug dosagesfor all animals were calculated according to the body weight (singleadministration (subcutaneously), administration dosage of 3 mg/kg and 1mg/kg for the siRNA conjugates (calculated based on the amount ofsiRNA), and administration volume of 5 mL/kg). Each siRNA conjugate wasadministered in the form of PBS aqueous solution. The concentration ofthe conjugate to be formulated was calculated based on the administereddosage and volume. The blood was taken from mouse orbital venous plexusbefore administration (marked as day −1) and on days 7, 14, 21, 28, 35,56, 70, 84, 98, 112, 126, 140, 154, and 168 after administration, andthe blood lipid level was measured for each time point by using the samemethod as in Experimental Example 4-2. The results are shown in FIGS.56A and 56B.

As can be seen from FIGS. 56A and 56B, the PBS blank control group andComparative Conjugate E1 negative control group showed no inhibitoryeffect on blood lipid at different time points after administration; incontrast, Conjugate E1 significantly reduced the contents of TG and CHO.For TG, the high dose group showed the maximum inhibition percentage of92.9% on day 7 after administration, and the low dose group showed themaximum inhibition percentage of 79.1% on day 21 after administration.The high dose group consistently showed an inhibition percentage of 55%or higher against TG over a period of up to 154 days after singleadministration; the low dose group consistently showed an inhibitionpercentage of 55% or higher against TG over a period of up to 98 daysafter single administration. For CHO, the high dose group showed themaximum inhibition percentage of 82.9% on day 14 after administration,and the low dose group showed the maximum inhibition percentage of 65.9%on day 14 after administration. The high dose group showed an inhibitionpercentage of 40% or higher against CHO over a period of up to 154 daysafter single administration, and the low dose group showed an inhibitionpercentage 40% or higher against CHO over a period of up to 56 daysafter single administration. FIGS. 56A and 56B indicated that ConjugateE1 allowed continuous, stable and efficient reduction effect on bloodlipid level within 168 days after single administration.

In other experiments, the same method above was used, except that: theconjugates to be administered were Conjugate E2 and ComparativeConjugate E2. Blood lipid test continued until day 70 afteradministration, and the results were shown in FIGS. 57A-57D.

FIGS. 57A-57B showed the inhibitory effects of Conjugate E2 at two doseson CHO at different time points after administration. The group of miceadministered with high dose showed the maximum inhibitory percentageagainst CHO of up to 74.3% on day 21 after single administration; andthe inhibitory percentage against CHO was consistently maintained at 50%or higher over a period of up to 70 days after administration. The groupof mice administered with low dose showed the maximum inhibitorypercentage against CHO of 59.5% on day 14 after administration.

FIGS. 57C and 57D showed the inhibitory effects of Conjugate E2 at twodoses on TG at different time points after administration. The group ofmice administered with high dose showed the maximum inhibitorypercentage against TG of up to 96.3% on day 14 after singleadministration; and the inhibitory percentage against TG wasconsistently maintained at 70% or higher over a period of up to 70 daysafter administration. The group of mice administered with low doseshowed the maximum inhibitory percentage against TG of 75.3% on day 14after administration.

As can be seen from FIGS. 57A-57D, Conjugate E2 consistently reduced theblood lipid level over a period of 70 days after single administration,and was obviously superior to Comparative Conjugate E2 at equal dose.

Experimental Example E5 Detection of the Inhibitory Efficiency of thesiRNA Conjugate Against the Expression Level of ANGPTL3 mRNA and theInhibitory Effect on Blood Lipid in Non-Human Primates In Vivo

12 Monkeys with metabolic syndrome (all male) were randomly divided intogroups, with 8 monkeys being administered with Conjugate E2, 4 monkeysbeing administered with Comparative Conjugate E1. Each siRNA conjugatewas dissolved in normal saline for injection to have a drugconcentration of 100 mg/ml (calculated based on the amount of siRNA).The drug dosages for all animals were calculated according to the bodyweight. A single dose was administered subcutaneously, with the dosageof 9 mg/kg, the injection amount of 0.09 mL/kg and the administrationvolume of no more than 2 ml for each administration site.

The blood was taken from the vein once a week during three weeks beforeadministration to measure indicators such as the blood lipid level,liver function, and blood routine examination. These indicators werere-measured respectively on days 7, 14, 21, 28 and 35 afteradministration.

The normalized blood lipid level=(1−the blood lipid content in the testgroup after administration/the blood lipid content in the test groupbefore administration)×100%. The blood lipid refers to total cholesterolor triglyceride.

The blood lipid content before administration is the mean value of theblood lipid during 3 weeks before administration, and is a baselinevalue marked as DO. The inhibitory effects against blood lipids areshown in FIGS. 58A and 58B.

FIGS. 58A and 58B showed that Conjugate E2 resulted in the maximuminhibition percentage of 68% against TG and the maximum inhibitionpercentage of 30% against CHO on day 28 after single administrationcompared with that before administration.

In the very day of administration (marked as before administration) andon day 28 after administration, a Percutanous transshepatic biopsy wasperformed to measure the mRNA expression level of the ANGPTL3 in theliver tissue. The expression was measured by real-time fluorescent qPCRusing the same method as described in Experimental Example 4-1, exceptthat the detection primers are different. The detection primers usedherein are shown in Table 11E. The inhibition percentage against ANGPTL3mRNA is shown in FIG. 58C.

TABLE 11E Sequences of the primers Nucleotide Genes SEQ ID NO.sequence(5′ → 3′) Monkey ANGPTL3 441 CTGGTGGTGGCATGATGAGT 442CTCTTCTCCGCTCTGGCTTAG Monkey GAPDH 443 GGGAGCCAAAAGGGTCATCA 444CGTGGACTGTGGTCATGAGT

FIG. 58C showed that Conjugate E2 resulted in an inhibition percentageof up to 83% against ANGPTL3 mRNA on day 28 after single administrationcompared with that before administration.

Further indicators were measured at each time point afteradministration, no abnormal changes in the blood platelet,glutamic-pyruvic transaminase and glutamic oxalacetic transaminase werefound, indicating that Conjugate E2 had relatively good safety; and noobvious toxic side effect was observed.

As can be seen from FIG. 58A-58C, Conjugate E2 showed the effects ofsignificantly reducing blood lipid level and inhibiting ANGPTL3 geneexpression in non-human primate, and at the same time exhibitedrelatively good safety.

The above results indicated that the siRNAs and conjugates provided bythe present invention can effectively inhibit the expression of ANGPTL3mRNA in liver and reduce the content of total cholesterol ortriglyceride in blood, and thus can prevent and/or treat blood lipidabnormalities and have good clinical application prospect.

Hereinbelow, an experiment for verifying the effects of the siRNAconjugates shown in Table 4F is illustrated.

Experimental Example F1 this Experiment Investigates the InhibitoryActivity In Vitro of the siRNA Conjugates of the Present InventionExperimental Example F1-1 On-Target Activity in In Vitro psiCHECK System

In this experimental example, Conjugate F20 was investigated in in vitropsiCHECK system for on-target activity. Specifically, Conjugate F20 wastested for the activity of targeting completely matching target sequence(of which the nucleotide sequence is completely complementary with thefull length nucleotide sequence of the antisense strand of theconjugate).

Conjugate F20 was tested using the method as described in ExperimentalExample A1-6. The results are shown in FIG. 59 , indicating thatConjugate F20 has good inhibitory activity in vitro.

Experimental Example F1-2 Measurement of IC₅₀ in In Vitro psiCHECKSystem

This experimental example investigates the IC₅₀ value of Conjugate F1 inin vitro psiCHECK system.

The on-target plasmid of Conjugate F1 was constructed using the samemethod as described in Experimental Example F1-1. The finalconcentration of Conjugate F1 (calculated based on the concentration ofsiRNA) was double diluted from 1 nM to 0.001 nM to give 11concentrations. The dose-response curves were plotted by the activityresults measured at different siRNA concentrations, and the curves werefitted using function log(inhibitor) vs. response-Variable slope ofGraphpad 5.0 software. The IC₅₀ value of Conjugate F1 was calculatedbased on the dose-response curves with the formula below:

$Y = {{Bot} + \frac{{Top} - {Bot}}{1 + \text{?}}}$?indicates text missing or illegible when filed

wherein

Y is the expression level of remaining mRNA,

X is the logarithm of the concentration of transfected siRNA,

Bot is the Y value at the bottom of the steady stage,

Top is the Y value at the top of the steady stage,

Log IC₅₀ is the X value at which Y is median value between the bottomand the top of the steady stage, and HillSlope is the slope of thecurve.

It was thus calculated that Conjugate F1 had an IC₅₀ value of 0.0174 nMin in vitro psiCHECK system, indicating that the siRNA conjugate of thepresent disclosure has higher activity in intro.

Experimental Example F1-3 Measurement of IC50 in In Vitro Cell Lines

In this experimental example, the inhibitory efficiency of Conjugate F2against the expression level of APOC3 mRNA in in vitro Huh 7 cells wasinvestigated.

Conjugate F2 was transfected to Human hepatoma cell lines Huh7 by usingLipofectamine™ 2000. The final concentration of siRNA conjugate wasdiluted 3-fold from 3 nM to 0.004 nM to give 7 concentrations, 2replicate wells per concentration.

The expression levels of APOC3 mRNAs in Huh 7 cells transfected withConjugate F2 at various concentrations were measured by PCR(Quantitative Real-Time PCR), respectively. Specific steps were asfollows: 24 hours after cultivation of transfected cells, the total RNAwas extracted and obtained by using Trizol (Thermo Fisher) according tothe standard procedure for total RNA extraction; 1 μg of the total RNAwas individually extracted and reverse transcribed into cDNA by usingreverse transcription kit (Promega, Cat No. A3500) according to theinstruction thereof. The expression level of APOC3 mRNA was detectedbased on the template cDNA according to the steps described in theinstruction by using 2×Ultra SYBR Mixture (with ROX) (Beijing CowinBiosicences Co., Ltd, Cat No. CW 0956). Therein, the PCR primers of3-actin for amplifying APOC3 and as an internal control gene are shownin Table 5F.

TABLE 5F  Sequences of the primers for detection Genes Upstream PrimersDownstream Primers Human 5′-GTGACCGATGGCT 5′-ATGGATAGGCAGGT APOC3TCAGTTC-3′ GGACTT-3′ (SEQ ID NO: 445) (SEQ ID NO: 446) Human5′-CCAACCGCGAGAA 5′-CCAGAGGCGTACA β-actin GATGA-3′ GGGATAG-3′(SEQ ID NO: 447) (SEQ ID NO: 448)

The expression level of APOC3 mRNA was calculated by the followingequation:

the expression level of APOC3 mRNA=[(the expression level of APOC3 mRNAin the test group/the expression level of β-actin mRNA in the testgroup)/(the expression level of APOC3 mRNA in the control group/theexpression level of β-actin mRNA in the control group)]×100%.

Therein, Huh7 cells individually treated with Conjugate F2 at variousconcentrations were used in the test groups, and Huh 7 cells untreatedwith Conjugate F2 were used in the control group.

The IC₅₀ value was calculated by the same method as in ExperimentalExample F1-2 according to the measured inhibition percentages ofConjugate F2 at different concentrations against the expression level ofAPOC3 mRNA. The IC₅₀ value of Conjugate F2 in in vitro Huh7 cells wasobtained to be 0.0085 nM, suggesting that the siRNA conjugate of thepresent disclosure has higher activity in intro.

Experimental Example F2 this Experimental Example Illustrated theInhibitory Efficiency of the siRNA Conjugate of the Present InventionAgainst the Expression Level of APOC3 mRNA In Vivo Experimental ExampleF2-1 this Experimental Example Investigated the Inhibition Percentage ofConjugate F1 Against the Expression Level of APOC3 mRNA in Liver Tissueof Human APOC3 Transgenic Mice In Vivo

Human APOC3 transgenic mice (B6; CBA-Tg(APOC3)3707Bres/J) were randomlydivided into groups based on TG content>2 mmol/L (5 mice in each group).Conjugate F1, Comparative Conjugate F1 and Normal saline (NS) wereindividually administered to the mice in each group. The drug dosagesfor all animals were calculated according to the body weight (singleadministration (subcutaneously), two administration dosage of 1 mg/kgand 0.1 mg/kg for the siRNA conjugates (calculated based on the amountof siRNA)). Each conjugate was administered at the concentrations of 0.2mg/mL and 0.02 mg/mL in the form of 0.9 wt % NaCl aqueous solution andthe administration volume of 5 mL/kg. The mice were sacrificed on day 14after administration. The liver was collected and kept with RNA later(Sigma Aldrich), and the liver tissue was homogenized with a tissuehomogenizer. Then the total RNA was extracted and obtained by usingTrizol according to the standard procedure for total RNA extraction.

The expression level of APOC3 mRNA in liver tissue was measured by thereal-time fluorescent qPCR method as described in Experimental ExampleF1-3. In this fluorescent qPCR method, (3-actin gene was used as aninternal control gene, the expression levels of APOC3 and β-actin weremeasured by using primers for APOC3 and β-actin, respectively.

The sequences of primers for detection are shown in Table 6F.

TABLE 6F Genes Upstream Primers Downstream Primers Human 5′-GTGACCGATGG5′-ATGGATAGGCAG APOC3 CTTCAGTTC-3′ GTGGACTT-3′ (SEQ ID NO: 449)(SEQ ID NO: 450) Mouse 5′-AGCTTCTTTGC 5′-TTCTGACCCATTC β-actinAGCTCCTTCGTTG-3′ CCACCATCACA-3′ (SEQ ID NO: 451) (SEQ ID NO: 452)

The inhibition percentage of the conjugate against APOC3 mRNA wascalculated according to the equation:

the inhibition percentage=(the expression level of APOC3 mRNA in thetest group/the expression level of β-actin mRNA in the test group)/(theexpression level of APOC3 mRNA in the control group/the expression levelof β-actin mRNA in the control group)×100%,

wherein the control group was a group of control mice administered withNS in this experiment and each test group was a group of miceadministered with different siRNA conjugates, respectively. The resultswere shown in FIG. 60 .

The results indicated that Conjugate F1 showed inhibitory effect onhuman APOC3 gene in transgenic mice.

Experimental Example F2-2 this Experimental Example Investigated theInhibition Percentage of Conjugate F1 Against the Expression Level ofAPOC3 mRNA in Liver Tissue of Cynomolgus Monkey In Vivo, and the Effecton Blood Lipid Level

CTI Biotechnology (Suzhou) Co., Ltd. was authorized to perform thisexperimental example. Cynomolgus monkeys (body weight: 2-4 kg, age: 3-5years old) were randomly divided into two groups, one male and onefemale per group. Conjugate F1 and Comparative Conjugate F2 wereadministered individually. The drug dosages for all animals werecalculated according to the body weight (single administration(subcutaneously), administration dosage of 3 mg/kg for the siRNAconjugates (calculated based on the amount of siRNA), in the form of 3mg/ml in the form of NaCl solution for injection (Shandong KelunPharmaceutical Co., Ltd.), and administration volume of 1 mL/kg). Thevery day on which the first dose is administered is defined as Test Day1 (D1), and one day before administration is defined as Day 0 (DO).

The blood samples were taken from the vein of the animals beforeadministration and on day 7, 14, 21 and 28 after administration. Thecontents of the substances to be detected in serum were measured at eachtime point. The substances to be detected include blood lipids (totalcholesterol (CHO) and triglyceride (TG)), and transaminases (glutamicpyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST).The substances to be detected were normalized. The inhibition percentageof each substance to be detected was calculated by the equation:

the inhibition percentage=(1−the content of the substance to be detectedin the test group after administration)/the content of the substance tobe detected in the test group before administration)×100%,

wherein the inhibition percentage against triglyceride (TG) is shown inTable 7F.

TABLE 7F Inhibition percentage against Content of TG (mmol/L) TG (%)Conjugate Time point D0 D7 D14 D21 D28 D7 D14 D21 D28 Conjugate 1 Male M0.88 0.32 0.35 0.42 0.3 63.6 60.2 52.3 65.9 Female F 0.75 0.31 0.4 0.520.23 58.7 46.7 30.7 69.3

Transaminase content was measured at each detection point afteradministration, and no abnormality on liver function was found.

The animals were sacrificed on day 28 after administration, and theliver was collected. No abnormalities were found in gross anatomy. RNAwas extracted from liver tissue by the same method as that described inExperimental Example F2-1, and the expression level of APOC3 mRNA inliver was measured. The sequences of the primers for detection are shownin Table 8F.

TABLE 8F Genes Upstream Primers Downstream Primers Monkey 5′-TTGAACCCTGA5′-CGGTAGGAGG APOC3 GGCCAAACC-3′ GCACTGAGAA-3′ (SEQ ID NO: 453)(SEQ ID NO: 454) Monkey 5′-GGGAGCCAAA 5′-CGTGGACTGTG GAPDH AGGGTCATCA-3′GTCATGAGT-3′ (SEQ ID NO: 455) (SEQ ID NO: 456)

The expression level of APOC3 mRNA can be measured by real-timefluorescent qPCR. Relative to the Comparative Conjugate F2, Conjugate F1resulted in an inhibition percentage of 55.3% against APOC3 mRNA in thefemale animals and an inhibition percentage of 78.5% against APOC3 mRNAin the male animals.

This experiment indicated that Conjugate F1 also exhibited significantlyinhibitory effect on APOC3 gene in non-human primate and showedsignificantly inhibitory effect on TG in serum, and at the same time noabnormalities on liver function were observed.

Experimental Example F3 this Experiment Investigated the Effects of thesiRNA Conjugate of the Present Invention on Blood Lipid Content In VivoExperimental Example F3-1 this Experiment Investigated the Effects ofConjugate F1 on the Contents of Total Cholesterol (CHO) and Triglyceride(TG) in Serum of Human APOC3 Transgenic Mice In Vivo

Human APOC3 transgenic mice (B6; CBA-Tg(APOC3)3707Bres/J) with TGcontent each being >2 mmol/L were randomly divided into groups (7 micefor each group): (1) NS control group; (2) Conjugate F1 3 mg/kg group;(3) Conjugate F1 1 mg/kg group. The drug dosages for all animals werecalculated according to the body weight (single administration(subcutaneously), in form of 0.9 wt % NaCl aqueous solution containing0.6 mg/ml and 0.2 mg/ml the siRNA conjugate, and administration volumeof 5 mL/kg).

The blood was taken from mouse orbital venous plexus beforeadministration (marked as day 0) and on days 7, 14, 21, 28, 35, 42, 49,63, 77, 91, 112, 133, 147, 154, 161, 175 and 189 after administrationrespectively. The contents of total cholesterol (CHO) and triglyceride(TG) in serum were measured at each time point.

About 100 μl orbital blood was taken each time, and the serum was noless than 20 μl after centrifugation. The contents of total cholesterol(CHO) and triglyceride (TG) in serum were further measured by using aPM1P000/3 full-automatic serum biochemical analyzer (SABA, Italy).

The normalized blood lipid level=(the blood lipid content in the testgroup after administration/the blood lipid content in the test groupbefore administration)×100%.

The inhibition percentage against blood lipid level=(1−the blood lipidcontent in the test group after administration/the blood lipid contentin the test group before administration)×100%. Blood lipid refers tototal cholesterol (CHO) or triglyceride (TG).

The measured results are shown in FIGS. 61A and 61B.

As can be seen from FIGS. 61A and 61B, Conjugate F1 showed the effect ofsignificantly reducing the contents of TG and CHO in mouse serum atdifferent time points after administration over a period of up to 189days, indicating that the conjugate has stable and effective inhibitionagainst the expression of APOC3 gene over a longer time period.

Experimental Example F3-2 this Experimental Example Investigated theEffects of Conjugate F2 on the Contents of Total Cholesterol (CHO) andTriglyceride (TG) in Human APOC3 Transgenic Mice Serum In Vivo

Detection was performed according to the same method as described inExperimental Example F3-1, except that: 8 mice for each group, theconjugate to be administered was Conjugate F2; five doses (0.1, 0.3, 1,3 and 9 mg/kg) were individually administered; the administration volumeremained unchanged and thus the concentration of conjugate solution wascorresponding adjusted; the test continued until day 112. The resultsare shown in FIGS. 62A and 62B.

As can be seen from the results of FIGS. 62A and 62B, Conjugate F2significantly reduced the contents of TG and CHO in transgenic mice overa period of up to 112 days, and there was obvious dose-dependentresponse in the reduction effect.

Experimental Example F3-3 this Experimental Example Compared the Effectsof Conjugates F1-F3 on the Contents of Total Cholesterol (CHO) andTriglyceride (TG) in Human APOC3 Transgenic Mice Serum In Vivo

The contents of total cholesterol (CHO) and triglyceride (TG) in mouseserum were measured using the same method as that in ExperimentalExample F3-1, except that: 6 mice for each group, Conjugates F1, F2 andF3 and Comparative Conjugate F2 were individually administered; twodoses (1 mg/kg and 3 mg/kg) were administered for each conjugate; theadministration volume remained unchanged and thus the concentration ofconjugate solution was correspondingly adjusted; the test continueduntil day 112. The results are shown in FIGS. 63A-63D.

As can be seen from the results of FIGS. 63A-63D, the Conjugates F1-F3of the present disclosure at different doses showed the effect ofconsistently reducing the blood lipid level in the transgenic mice in upto 112 days, and this reduction effect as a whole was superior to thatof Comparative Conjugate F2.

The above results indicated that the conjugates provided by the presentinvention effectively inhibited the expression of APOC3 mRNA in liverand reduced the content of total cholesterol or triglyceride in blood,and thus can prevent and/or treat blood lipid abnormalities and havegood clinical application prospect.

Hereinbelow, an experiment for verifying the effects of the siRNAconjugates shown in Table 4G was illustrated.

Experimental Example G1 this Experimental Example Indicated that thesiRNA Conjugate of the Present Disclosure not Only has Higher ActivityIn Vitro, but Also Shows Low Off-Target Effect

HEK293A cells used in this experimental example were provided by NucleicAcid Technology Laboratory, Institute of Molecular Medicine, PekingUniversity, and cultured in DMEM complete media (Hyclone company)containing 20% fetal bovine serum (FBS, Hyclone company), 0.2 v %Penicillin-Streptomycin (Gibco, Invitrogen company) at 37° C. in anincubator containing 5% CO₂/95% air.

In this experimental example, Conjugate G2 was investigated in in vitropsiCHECK system for the on-target activity and off-target effect.Specifically, Conjugate G2 was tested for the activity of targetingcompletely matching target sequence (of which the nucleotide sequence iscompletely complementary with the full length nucleotide sequence of thesense/antisense strand of Conjugate G2) or targeting seed regionmatching target sequence (of which the nucleotide sequence iscomplementary with the nucleotide sequence of positions 1-8 of thesense/antisense strand of Conjugate G2).

Conjugate G2 was tested according to the method described inExperimental Example A1-6, except that four target sequences wereconstructed based on the sequences of Conjugate G2. For the on-targetplasmid GSCM, the final concentration of Conjugate G2 (calculated basedon the concentration of siRNA) was double diluted from 1 nM to 0.000977nM to give 11 concentrations; and for the other 3 off-target plasmids,the final concentration of Conjugate G2 was 4-fold diluted from 10 nM to0.000038 nM to give 10 concentrations.

For GSCM, the IC₅₀ value of Conjugate G2 was 0.0513 nM (R²=0.9911); forPSCM, GSSM, and PSSM, Conjugate G2 showed no obvious inhibitory effectat each siRNA concentration, indicating that the siRNA conjugate of thepresent disclosure not only has higher activity in vitro, but also showslow off-target effect.

Experimental Example G2 this Experimental Example Illustrated Stabilityof the siRNA Conjugates of the Present Disclosure in the Lysosome LysateIn Vitro

1) Detection of the Stability in Murine Lysosome Lysate

Preparation of test samples treated with the lysosome lysate: 6 μl eachof Conjugate G2 and Comparative siRNA1 (20 μM) were individually mixedwell with 27.2 μL of sodium citrate aqueous solution (pH 5.0), 4.08 μLof deionized water and 2.72 μL of murine lysosome lysate (Rat LiverTritosomes, purchased from Xenotech Inc., Cat No. R0610.LT, Lot No.1610069, at a final concentration of acid phosphatase of 0.2 mU/μL), andincubated at a constant temperature of 37° C. 5 μL mixed solutions weretaken at each time point of 0 h, 1 h, 2 h, 4 h, 6 h, and 24 h,respectively, added to 15 μL of 9 M urea aqueous solution fordenaturation, and added with 4 μL of 6× loading buffer (purchased fromSolarbio Inc., Cat No. 20160830), then immediately cryopreserved in a−80° C. freezer to quench the reaction. 0 h represents the moment whenthe sample was taken immediately after the samples to be tested aremixed well with the lysosome lysate.

Preparation of control samples untreated with the lysosome lysate: 1.5μL each of the Conjugate G2 and Comparative siRNA1 (20 μM) at equalmoles was mixed well with 7.5 μL of sodium citrate aqueous solution (pH5.0) and 1 μL of deionized water, added to 30 μL of 9 M urea solutionfor denaturation, and added with 8 μL of 6×loading buffer, thenimmediately cryopreserved in a −80° C. freezer to quench the reaction.The control sample for each conjugate is marked as M to be compared withthe electrophoresis results of the sample.

16 wt % of non-denatured polyacrylamide gel was prepared. 20 μL each ofthe test sample and the control sample described above was loaded ontothe gel to perform electrophoresis for 10 minutes under 20 mA constantcurrent and then for 30 minutes under 40 mA constant current. Afterfinishing the electrophoresis, the gel was placed on a shaker andstained with Gelred dye (BioTium, Cat No. 13G1203) for 10 minutes. Thegel was subjected to imaging, observation and photocopying. The resultsare shown in FIG. 64 .

2) Detection of the Stability in Human-Originated Lysosome Lysate

The stability of Comparative siRNA1 and Conjugate G2 in the humanlysosome lysate was measured using the same method as in 1), except thatthe murine lysosome lysate was replaced with the human lysosome lysate(Human Liver Lysosomes, purchased from Xenotech Inc., Cat No. R0610.L,Lot No. 1610316). The results are shown in FIG. 65 .

The results of FIGS. 64 and 65 indicated that the siRNA conjugates ofthe present disclosure can remain undegraded for at least 24 hourseither in human lysosome lysate or murine lysosome lysate, showingsatisfactory stability.

Experimental Example G3 this Experimental Example Illustrated theInhibitory Efficiency of the siRNA Conjugates of the Present DisclosureAgainst the Expression Level of HBV mRNA in HBV Model Mice

1) In this experimental example, the inhibition efficiency of ConjugatesG1 and G2 against the expression level of HBV mRNA in HBV model miceC57BL/6J-Tg(Alb1HBV)44Bri/J was investigated.

The C57BL/6J-Tg (Alb1HBV) 44Bri/J mice used herein were purchased fromDepartment of Laboratory Animal Science, Peking University HealthScience Center. Conjugate G2 was formulated with 0.9% NaCl aqueoussolution into a solution with a concentration of 0.2 mg/ml (calculatedbased on the concentration of siRNA); and Conjugate 1 was formulatedwith 0.9% NaCl aqueous solution into solutions with the concentrationsof 0.2 mg/ml and 0.06 mg/ml (calculated based on the concentration ofsiRNA).

HBsAg content in mouse serum was measured using Hepatitis B VirusSurface Antigen Assay Kit (Enzyme-linked Immunosorbent Assay, ELISA)(Shanghai Kehua Bio-engineering Co., Ltd.). Mice with S/COV>10 wereselected and randomly divided into two groups (all female, 6 mice ineach group) and respectively numbered as the control group and the testgroup. Each group of the animals was administered subcutaneously withthe respective drugs on day 1 in the volume of 5 mL/kg. The drug dosagesfor all animals were calculated according to the body weight. Therein,mice in the control group were administered with normal saline; and micein the test group were administered with Conjugate G2, with theadministration dose being 1 mg/kg. All the animals were sacrificed onday 28 after administration, and were subjected to gross anatomy toobserve whether the organs in the body were diseased. The diseasedtissues by visual observation were kept in 10% formalin for furtherpathological observation. The liver was collected and kept with RNAlater (Sigma Aldrich), the liver tissue was homogenized with a tissuehomogenizer, and then total RNAs were extracted and obtained by usingTrizol according to the standard procedure for total RNA extraction.

The expression level of HBV mRNA in liver tissue was measured byreal-time fluorescent qPCR. Specifically, the extracted total RNA wasreverse transcribed into cDNA by using ImProm-II™ reverse transcriptionkit (Promega) according to the instruction, and then the inhibitoryefficiency of siRNAs against the expression of HBV mRNA in liver tissuewas measured by using the fluorescent qPCR kit (Beijing CowinBiosicences Co., Ltd). In this fluorescent qPCR method, β-actin gene wasused as an internal control gene, the HBV and 0-actin were detected byusing primers for HBV and β-actin, respectively.

Sequences of primers for detection are shown in Table 5A.

In this fluorescent qPCR method, the inhibitory activity of siRNA wasexpressed as the inhibition percentage against HBV mRNA and calculatedby the equation:

the inhibition percentage against HBV mRNA=(1−the remaining expressionof HBV gene)×100%;

The remaining expression of HBV gene=(the copy number of HBV gene in thetest group/the copy number of β-actin gene in the test group)/(the copynumber of HBV gene in the control group/the copy number of β-actin genein the control group)×100%.

The results are shown in Table 6G below.

The inhibitory efficiency in vivo of Conjugate 1 at different doses(n=5) against the expression level of HBV mRNA was detected using thesame method. The results are shown in Table 6G below.

TABLE 6G Inhibition percentage against HBV Dose P mRNA in siRNAConjugate NO. (mg/kg) liver (%) Conjugate G2 L10-siHB3M1SP 1 77.41Conjugate G1 L10-siHB3M1SVP 1 88.27 Conjugate G1 L10-siHB3M1SVP 0.357.95

As can be seen from the results shown above, all conjugates of thepresent disclosure showed high inhibitory activity against HBV mRNA inmice in vivo, indicating good in vivo delivery efficiency of the siRNAconjugates of the present disclosure.

2) The inhibitory efficiency in vivo of Conjugates G3-G20 against HBVmRNA was detected using the same method as in 1). It can be expectedthat Conjugates G3-G20 also exhibit higher inhibitory activity againstHBV mRNA.

Experimental Example G4 This experiment illustrated a test about therelationship between time and the inhibitory efficiency of the siRNAconjugates of the present disclosure against the expression levels ofHBsAg, HBeAg and HBV DNA in HBV model mice serum.

HBV model mice C57B/6N-Tg (1.28 HBV)/Vst (genotype A) used in thisexperimental example was purchased from Beijing Vitalstar BiotechnologyCo., Ltd. Conjugate G1 was formulated with 0.9% NaCl aqueous solutioninto solutions with the concentrations of 0.6 mg/ml and 0.2 mg/ml(calculated based on the concentration of siRNA).

The mice with HBsAg content in serum >10⁴ COI (half female, half male)were selected and randomly divided into three groups (6 mice in eachgroup), which are respectively numbered as the control group, thehigh-dose group and the low-dose group. The drugs were individuallyadministered subcutaneously to the mice in each group on day 1, with theadministration volume being 5 mL/kg. The drug dosages for all animalswere calculated according to the body weight. All the animals wereadministered before noon. If it is necessary to take the blood, theadministration would be conducted after the blood have been taken.Therein, the mice in the control group were injected with normal saline;and the mice in the test groups were injected with different doses ofConjugate G1: 3 mg/kg for the high-dose group and 1 mg/kg for thelow-dose group. The blood was taken from mouse orbital venous plexusbefore administration and on days 7, 13, 21, 28, 42, 56, 70, 84, 98,112, 126, 140, and 154 after administration, and HBsAg, HBeAg and HBVDNA levels in serum were measured for each time point.

About 100 μl orbital blood was taken each time; and the serum was noless than 20 μl after centrifugation, re-suspended with PBS to 500 μland delivered to Clinical Laboratory Center of Beijing DIAN Diagnosticsto measure the contents of HBsAg, HBeAg and HBV DNA in serum, which wereexpressed in COI, COI and IU/ml, respectively.

The normalized levels of the indicators to be measured (HBeAg, HBeAg andHBV DNA) were calculated according to the equation: the normalized levelof the indicator to be measured=(the remaining content of the indicatorto be measured after administration/the content of the indicator to bemeasured before administration)×100%.

The inhibition percentage of the indicator to be measured=(1−thenormalized level of the indicator to be measured)×100%.

The experimental data are expressed as X±SEM, and the data are analyzedwith Graphpad prism 5.0 statistical analysis software. The data areinitially tested for normal distribution and homogeneity of variance. Ifthe data meet normal distribution (p>0.2) and homogeneity of variance(p>0.10), then comparison among groups would be performed by LSD methodusing single-factor analysis of variance for multiple comparisons.P<0.05 is considered as being statistically significant. If the datafail to meet normal distribution and homogeneity of variance, comparionamong groups would be performed by Krushkal-Wallis H method forNon-parametric Test. If the results obtained by Krushkal-Wallis H testare statistically significant (p<0.05), pairwise comparisons amongmultiple groups would be conducted after rank transformation. P<0.05 isconsidered to be statistically significant.

The results are shown in FIGS. 66-68 .

As can be seen from FIG. 66 , the negative control group administeredwith normal saline showed no inhibitory effect on HBsAg at differenttime points after administration. In contrast, Conjugate G1 at the twodoses both showed excellent inhibitory effects on HBsAg at differenttime points after administration. For the high dose group, the maximuminhibitory percentage against HBsAg was up to 99.8% on day 13 aftersingle administration; the inhibitory percentage against HBsAg was stillmaintained at 90% or higher over a period of up to 84 days afteradministration; and until the end of the observation, the inhibitorypercentage against HBsAg was still up to 80.1%. For the low dose group,the maximum inhibitory percentage against HBsAg was 99.0% on day 13after administration; until the end of the observation on day 154, theinhibitory percentage against HBsAg was still up to 60.8%.

As can be seen from FIG. 67 , Conjugate G1 likewise inhibited theexpression of HBeAg, wherein the high-dose group showed an inhibitionpercentage of about 50% against HBeAg in serum on day 70 afteradministration; and until the end of the observation on day 154, theinhibitory efficiency against HBeAg was rebound to the level beforeadministration.

As can be seen from FIG. 68 , Conjugate G1 further efficiently inhibitedthe expression of HBV DNA and maintained higher inhibit ratio over anobservation period of up to 154 days. The high-dose group showed themaximum inhibition percentage of up to 99.2% against HBV DNA on day 13after single administration; the inhibitory percentage against HBV DNAwas still maintained at 90% or higher over a period of up to 84 daysafter administration; and until the end of the observation, theinhibitory percentage against HBV DNA was still up to 77.0%. Thelow-dose group showed the maximum inhibitory percentage against HBV DNAof up to 95.4% on day 13 after administration; until the end of theobservation on day 154, the inhibitory percentage against HBV DNA wasstill up to 79.4%.

The results described above indicated that the conjugates of the presentdisclosure showed stable and effective inhibition against the expressionof HBV gene over a longer time period, and in particular exhibitedlong-time and effective inhibition against surface antigen, showingexcellent effects.

Embodiments of the present disclosure are described above in detail, butthe present disclosure is not limited to the specific details of theabove-described embodiments. Various simple variations of the technicalsolution of the present disclosure can be made within the scope of thetechnical concept of the present disclosure, and these simple variationsare within the scope of the present disclosure.

It is to be noted that each of the specific technical features describedin the above embodiments can be combined in any suitable manner as longas no contradiction is caused. In order to avoid unnecessary repetition,the various possible combination manners are no longer described in thepresent disclosure.

In addition, the various different embodiments of the present disclosuremay also be carried out in any combination as long as it does notcontravene the idea of the present disclosure, which should also beregarded as the disclosure of the present disclosure.

1. A double-stranded oligonucleotide, which comprises a sense strand andan antisense strand, each nucleotide in the sense strand and antisensestrand being a modified nucleotide, wherein the sense strand comprises anucleotide sequence 1, and the antisense strand comprises a nucleotidesequence 2; the nucleotide sequence 1 and the nucleotide sequence 2 areboth 19 nucleotides in length and are at least partly reversecomplementary to form a double-stranded region; the nucleotide sequence2 is at least partly reverse complementary to a first nucleotidesequence segment, which refers to a segment of nucleotide sequence inthe target mRNA; in the direction from 5′ terminal to 3′ terminal, thenucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 arefluoro modified nucleotides, and each of the nucleotides at the otherpositions in the nucleotide sequence 1 is independently a non-fluoromodified nucleotide; the first nucleotide at 5′ terminal of thenucleotide sequence 2 is the first nucleotide at 5′ terminal of theantisense strand, the nucleotides at positions 2, 6, 14 and 16 of thenucleotide sequence 2 are fluoro modified nucleotides, and each of thenucleotides at the other positions in the nucleotide sequence 2 isindependently a non-fluoro modified nucleotide.
 2. The double-strandedoligonucleotide according to claim 1, wherein in the direction from 5′terminal to 3′ terminal, at least the nucleotides at positions 2-19 ofthe nucleotide sequence 2 are complementary to the first nucleotidesequence segment, or wherein in the direction from 5′ terminal to 3′terminal, the nucleotide at position 1 of the nucleotide sequence 2 is Aor U.
 3. The double-stranded oligonucleotide according to claim 1,wherein the sense strand further comprises nucleotide sequence 3, andthe antisense strand further comprises nucleotide sequence 4; eachnucleotide in the nucleotide sequence 3 and the nucleotide sequence 4 isindependently a non-fluoro modified nucleotide; the nucleotide sequence3 and the nucleotide sequence 4 are respectively 1-4 nucleotides inlength; the nucleotide sequence 3 and the nucleotide sequence 4 haveequal length and are substantially reverse complementary or completelyreverse complementary to each other; the nucleotide sequence 3 is linkedto the 5′ terminal of the nucleotide sequence 1; and the nucleotidesequence 4 is linked to the 3′ terminal of the nucleotide sequence 2;the nucleotide sequence 4 is substantially reverse complementary, orcompletely reverse complementary to a second nucleotide sequencesegment, which refers to a nucleotide sequence that is adjacent to thefirst nucleotide sequence segment in the target mRNA and has the samelength as the nucleotide sequence 4; “substantially reversecomplementary” refers to no more than 1 base mispairing between twonucleotide sequences; and “completely reverse complementary” refers tono mispairing between two nucleotide sequences.
 4. The double-strandedoligonucleotide according to claim 1, wherein the double-strandedoligonucleotide also comprises a nucleotide sequence 5; each nucleotidein the nucleotide sequence 5 is independently a non-fluoro modifiednucleotide; the nucleotide sequence 5 is 1-3 nucleotides in length andis linked to 3′ terminal of the antisense strand, thereby forming a 3′overhang of the antisense strand.
 5. The double-stranded oligonucleotideaccording to claim 1, wherein the nucleotide sequence 5 is 2 nucleotidesin length; and in the direction from 5′ terminal to 3′ terminal, thenucleotide sequence 5 is 2 consecutive thymidine deoxynucleotides or 2consecutive uridine nucleotides, or is completely reverse complementaryto a third nucleotide sequence segment, which refers to a nucleotidesequence that is adjacent to the first or second nucleotide sequencesegment in the target mRNA and has the same length as the nucleotidesequence
 5. 6. The double-stranded oligonucleotide according to claim 1,wherein each non-fluoro modified nucleotide is a methoxy modifiednucleotide, wherein the methoxy modified nucleotide refers to anucleotide formed by substituting the 2′-hydroxy of the ribose group ofthe nucleotide with a methoxy group.
 7. The double-strandedoligonucleotide according to claim 1, wherein in the double-strandedoligonucleotide, wherein at least one phosphate group is aphosphorothioate, and the phosphorothioate linkage exists in at leastone of the following positions: the position between the first andsecond nucleotides at 5′ terminal of the sense strand; the positionbetween the second and third nucleotides at 5′ terminal of the sensestrand; the position between the first and second nucleotides at 3′terminal of the sense strand; the position between the second and thirdnucleotides at 3′ terminal of the sense strand; the position between thefirst and second nucleotides at 5′ terminal of the antisense strand; theposition between the second and third nucleotides at 5′ terminal of theantisense strand; the position between the first and second nucleotidesat 3′ terminal of the antisense strand; and the position between thesecond and third nucleotides at 3′ terminal of the antisense strand. 8.The double-stranded oligonucleotide according to claim 1, wherein thenucleotide at 5′-terminal of the antisense strand is a 5′-phosphatenucleotide or a 5′-phosphate analogue modified nucleotide.
 9. Thedouble-stranded oligonucleotide according to claim 1, wherein thedouble-stranded oligonucleotide is an saRNA or an siRNA.
 10. Thedouble-stranded oligonucleotide according to claim 9, wherein the targetmRNA is one of the mRNAs corresponding to the following genes: ApoB,ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, andHCV.
 11. The double-stranded oligonucleotide according to claim 10,wherein, the nucleotide sequence 1 is a sequence shown by SEQ ID NO: 1,and the nucleotide sequence 2 is a sequence shown by SEQ ID NO: 2; orthe nucleotide sequence 1 is a sequence shown by SEQ ID NO: 3, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 4; or thenucleotide sequence 1 is a sequence shown by SEQ ID NO: 5, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 6; or thenucleotide sequence 1 is a sequence shown by SEQ ID NO: 7, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 8; or thenucleotide sequence 1 is a sequence shown by SEQ ID NO: 9, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 10; or thenucleotide sequence 1 is a sequence shown by SEQ ID NO: 11, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 12; or thenucleotide sequence 1 is a sequence shown by SEQ ID NO: 13, and thenucleotide sequence 2 is a sequence shown by SEQ ID NO: 14;(SEQ ID NO: 1) 5′-CmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3′(SEQ ID NO: 2) 5′-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGm-3′(SFQ ID NO: 3) 5′-UmGmCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm-3′(SEQ ID NO: 4) 5′-UmAfGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAm-3′(SEQ ID NO: 5) 5′-UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3′(SEQ ID NO: 6) 5′-UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAm-3′(SEQ ID NO: 7) 5′-CmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3′(SEQ ID NO: 8) 5′-UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGm-3′(SEQ ID NO: 9) 5′-GmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm-3′(SEQ ID NO: 10) 5′-UmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCm-3′(SEQ ID No: 11) 5′-CmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3′(SEQ ID No: 12) 5′-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGm-3′(SEQ ID No: 13) 5′-CmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′(SEQ ID No: 14) 5′-UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGm-3′

wherein C, G, U, and A represent the base components of the nucleotides;m represents that the nucleotide adjacent to the left side of the letterm is a 2′-methoxy modified nucleotide; f represents that the nucleotideadjacent to the left side of the letter f is a 2′-fluoro modifiednucleotide.
 12. A pharmaceutical composition, wherein the pharmaceuticalcomposition comprises the double-stranded oligonucleotide according toclaim 1 and a pharmaceutically acceptable carrier.
 13. Anoligonucleotide conjugate, comprising the double-strandedoligonucleotide according to claim 1 and a conjugation group conjugatedto the double-stranded oligonucleotide.
 14. The oligonucleotideconjugate according to claim 13, wherein the oligonucleotide conjugatehas a structure as shown by Formula (308):

wherein n1 is an integer of 1-3, and n3 is an integer of 0-4; m1, m2,and m3 independently of one another are an integer of 2-10; R₁₀, R₁₁,R₁₂, R₁₃, R₁₄, and R₁₅ independently of one another are H, or selectedfrom the group consisting of C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl and C₁-C₁₀alkoxy; R₃ is a group having a structure as shown by Formula (A59):

wherein, E₁ is OH, SH or BH₂; Nu is a double-stranded oligonucleotide;R₂ is a linear alkylene of 1 to 20 carbon atoms in length, wherein oneor more carbon atoms are optionally replaced with one or more groupsselected from the group consisting of: C(O), NH, O, S, CH═N, S(O)₂,C₂-C₁₀ alkylene, C₂-C₁₀ alkynylene, C₆-C₁₀ arylene, C₃-C₁₈heterocyclylene, and C₅-C₁₀ heteroarylene, and wherein R₂ optionally hasone or more substituents selected from the group consisting of C₁-C₁₀alkyl, C₆-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₁-C₁₀ haloalkyl, —OC₁-C₁₀ alkyl,—OC₁-C₁₀ alkylphenyl, —C₁-C₁₀ alkyl-OH, —OC₁-C₁₀ haloalkyl, —SC₁-C₁₀alkyl, —SC₁-C₁₀ alkylphenyl, —C₁-C₁₀ alkyl-SH, —SC₁-C₁₀ haloalkyl, halo,—OH, —SH, —NH₂, —C₁-C₁₀ alkyl-NH₂, —N(C₁-C₁₀ alkyl)(C₁-C₁₀ alkyl),—NH(C₁-C₁₀ alkyl), cyano, nitro, —CO₂H, —C(O)OC₁-C₁₀ alkyl, —CON(C₁-C₁₀alkyl)(C₁-C₁₀ alkyl), —CONH(C₁-C₁₀ alkyl), —CONH₂, —NHC(O)(C₁-C₁₀alkyl), —NHC(O)(phenyl), —N(C₁-C₁₀ alkyl)C(O)(C₁-C₁₀ alkyl), —N(C₁-C₁₀alkyl)C(O)(phenyl), —C(O)C₁-C₁₀ alkyl, —C(O)C₁-C₁₀ alkylphenyl,—C(O)C₁-C₁₀ haloalkyl, —OC(O)C₁-C₁₀ alkyl, —SO₂(C₁-C₁₀ alkyl),—SO₂(phenyl), —SO₂(C₁-C₁₀ haloalkyl), —SO₂NH₂, —SO₂NH(C₁-C₁₀ alkyl),—SO₂NH(phenyl), —NHSO₂(C₁-C₁₀ alkyl), —NHSO₂(phenyl), and —NHSO₂(C₁-C₁₀haloalkyl); each L₁ is independently a linear alkylene of 1 to 70 carbonatoms in length, wherein one or more carbon atoms are optionallyreplaced with one or more groups selected from the group consisting of:C(O), NH, O, S, CH═N, S(O)₂, C₂-C₁₀ alkylene, C₂-C₁₀ alkynylene, C₆-C₁₀arylene, C₃-C₁₈ heterocyclylene, and C₅-C₁₀ heteroarylene, and whereinL₁ optionally has one or more substituents selected from the groupconsisting of: C₁-C₁₀ alkyl, C₆-C₁₀ aryl, C₅-C₁₀ heteroaryl, C₁-C₁₀haloalkyl, —OC₁-C₁₀ alkyl, —OC₁-C₁₀ alkylphenyl, —C₁-C₁₀ alkyl-OH,—OC₁-C₁₀ haloalkyl, —SC₁-C₁₀ alkyl, —SC₁-C₁₀ alkylphenyl, —C₁-C₁₀alkyl-SH, —SC₁-C₁₀ haloalkyl, halo, —OH, —SH, —NH₂, —C₁-C₁₀ alkyl-NH₂,—N(C₁-C₁₀ alkyl)(C₁-C₁₀ alkyl), —NH(C₁-C₁₀ alkyl), cyano, nitro, —CO₂H,—C(O)OC₁-C₁₀ alkyl, —CON(C₁-C₁₀ alkyl)(C₁-C₁₀ alkyl), —CONH(C₁-C₁₀alkyl), —CONH₂, —NHC(O)(C₁-C₁₀ alkyl), —NHC(O)(phenyl), —N(C₁-C₁₀alkyl)C(O)(C₁-C₁₀ alkyl), —N(C₁-C₁₀ alkyl)C(O)(phenyl), —C(O)C₁-C₁₀alkyl, —C(O)C₁-C₁₀ alkylphenyl, —C(O)C₁-C₁₀ haloalkyl, —OC(O)C₁-C₁₀alkyl, —SO₂(C₁-C₁₀ alkyl), —SO₂(phenyl), —SO₂(C₁-C₁₀ haloalkyl),—SO₂NH₂, —SO₂NH(C₁-C₁₀ alkyl), —SO₂NH(phenyl), —NHSO₂(C₁-C₁₀ alkyl),—NHSO₂(phenyl), and —NHSO₂(C₁-C₁₀ haloalkyl);

represents the site where a group is linked to the rest of the molecule;M₁ represents a targeting group.
 15. The oligonucleotide conjugateaccording to claim 14, wherein each L₁ is independently selected fromthe group consisting of groups A1-A26, and any combination thereof:

wherein each j1 is independently an integer of 1-20; each j2 isindependently an integer of 1-20; R′ is a C₁-C₁₀ alkyl; Ra is selectedfrom the group consisting of A27-A45:


16. The oligonucleotide conjugate according to claim 15, wherein L1 isselected from A1, A4, A5, A6, A8, A10, A11, and A13, and any connectioncombination thereof, or L1 is selected from the connection combinationsof at least two of A1, A4, A8, A10, and All; or L1 is selected from theconnection combinations of at least two of A1, A8 and A10.
 17. Theoligonucleotide conjugate according to claim 14, wherein L1 is 3 to 25atoms in length, or L1 is 4 to 15 atoms in length.
 18. Theoligonucleotide conjugate according to claim 14, wherein n1 and n2independently of one another are an integer of 1 or 2; or n1+n3=2-3. 19.The oligonucleotide conjugate according to claim 14, wherein m1, m2 andm3 independently of one another are an integer of 2-5, or m1=m2=m3. 20.The siRNA conjugate according to claim 14, wherein each of the targetinggroups is selected from ligands capable of binding to cell surfacereceptor; or each of the targeting groups is selected from ligands thathave affinity to the asialoglycoprotein receptor (ASGP-R) on the surfaceof mammalian hepatocyte; or at least one or each of the targeting groupsis galactose or N-acetylgalactosamine (GalNAc).
 21. The oligonucleotideconjugate according to claim 14, wherein R10, R11, R12, R13, R14, andR15 independently of one another are selected from H, methyl or ethyl.22. The oligonucleotide conjugate according to claim 14, wherein R2forms an amide bond with the N atom on the nitrogenous backbone; or R2is B5, B6, B5′ or B6′:

wherein

represents the site where a group is linked to the rest of the molecule;q2 is an integer of 1-10.
 23. The oligonucleotide conjugate according toclaim 14, wherein the conjugate has a structure as shown by Formula(403), (404), (405), (406), (407), (408), (409), (410), (411), (412),(413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):


24. A method for treating a pathological condition or disease caused byabnormal gene expression, comprising administering an effective amountof the double-stranded oligonucleotide according to claim 1 and/or theconjugate thereof, to a subject in need thereof.
 25. The methodaccording to claim 24, wherein the gene is selected from the groupconsisting of the gene of hepatitis B virus, the gene ofangiopoietin-like protein 3, and the gene of apolipoprotein C3.
 26. Themethod according to claim 24, wherein the disease is selected fromchronic diseases, inflammations, fibrotic diseases, proliferativediseases and dyslipidemia.
 27. A method for inhibiting the expression ofa gene, comprising contacting an effective amount of the double-strandedoligonucleotide according to claim 1 and/or the conjugate thereof.